Difference between revisions of "Part:BBa K5160003"

Revision as of 11:31, 29 September 2024

Thaumatin Ⅱ

Overview

In order to provide people with a safe sweetener, our project hopes to propose a method for the efficient production of Thaumatin. Thaumatin comes from a tropical fruit in Africa and has been certified by the FDA and GRAS as a safe and reliable sugar substitute. Thaumatin binds to receptors on the human tongue to induce a sweet taste. This process produces almost no calories. At the same time, It can be fully digested by the human body and produce no side effects. This shows that Thaumatin is a very safe and reliable sweetener, which makes it an excellent choice for sugar reduction and control. About the engineering cycle and iteration of Thaumatin. Please go to the engineering section to find out more about the engineering cycle and the iteration of Thaumatin production.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NotI site found at 109
Illegal NotI site found at 146
Illegal NotI site found at 248
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 142
Illegal NgoMIV site found at 308
1000
COMPATIBLE WITH RFC[1000]

Usage and biology

Thaumatin is derived from the aril of the tropical plant Thaumatococcus daniellii (Benth). Bamboo yam contains two types of Thaumatin: Thaumatin I and Thaumatin II. One of them, Thaumatin II, has a higher sweetness. It consists of 207 amino acids, and the amino acids are coiled and folded to form eight disulfide bonds. As a result, the protein structure of Thaumatin is stable, thermally and acid-stable.

Thaumatin belongs to the family of five pathogenesis-related proteins (PR5), which are characterized by significant antifungal as well as phytopathogenic activity. Thus, in bamboo yam, the main function of Thaumatin is to protect seeds and fruits against abiotic stresses. After discovering the sweet flavor characteristics of Thaumatin, people have developed it and used it in various foods.

Thaumatin binds to sweetness receptors T1R2 and T1R3 on the human tongue, activating G-protein-coupled receptors on the membrane surface, generating an electrical signal that is transmitted to the cerebral cortex, causing sweetness perception. As a protein, Thaumatin consists of common amino acids and can be fully digested and absorbed by the body without generating calories.

Fig 1. Origin and protein structure of Thaumatin.

Design

Sequence identification

We found the original record of heterologous expression of Thaumatin from the literature. However, the recorded fragment was incomplete, so we blasted the incomplete sequence in NCBI to obtain the complete cds sequence. Finally, we obtained the complete sequence of Thaumatin II on the whole genome.

Fig 2. Nucleotide BLAST results on NBCI.

Chassis selection

Because of the complex structure of Thaumatin, we hoped to find a suitable chassis to express it efficiently.

The successful expression of the protein is related to its size, but it mainly depends on whether the protein expressed in the chassis can be properly transcribed, translated and folded.

Firstly, we tried to express Thaumatin and Brazzein in E. coli by constructing expression vectors and transformed them into E. coli, then induced the expression with IPTG and tested the protein level. Both Thaumatin and Brazzein have the special structure of disulfide bond, and the prokaryotic expression system has natural defects, which is not able to fold proteins correctly to form a complex structure, so the protein expression results were not satisfactory.

Fig 3. The expression of Thaumatin in the prokaryotic system.

Considering the practical application of sugar substitutes as a food additive, and the fact that fermentation production requires the complex process of protein purification, which may introduce safety concerns, we aim to simplify the Thaumatin production process by eliminating the need for purification steps. Thus, we have turned our focus to a fruit commonly consumed in daily life—tomatoes.

Pathway design

To validate the feasibility of the tomato platform, we need a quick testing method. Therefore, we aim to achieve this goal through transient expression. Many studies have shown that transient infection can be accomplished by Agrobacterium dipping or by viral vector leaf injection. Comparing the two methods, we found that Agrobacterium dipping struggles to maintain traits. A more critical issue is that it is difficult for it to complete infection in tomato fruits. In contrast, viral expression vectors can maintain traits for a longer period, which is advantageous for testing in tomato fruits. Therefore, we chose to use viral expression vectors for this step of validation.

TRV is a double-stranded RNA virus with a genome consisting of RNA1 and RNA2. With the reverse transcriptase, the TRV genome is reverse transcribed into cDNA in vitro. Specific modifications and cloning of the cDNA transform it into a non-toxic vector that retains both self-replication ability and the ability to express foreign genes, known as TRV1 and TRV2. The TRV1 vector usually contains genes encoding RNA-dependent RNA polymerase (RdRp), movement protein (MP), and a 16 kDa protein. It serves as an auxiliary viral vector to assist in the replication and spread of the virus within the plant. TRV2 contains the gene encoding the viral coat protein, and an artificially inserted MCS sequence is used for cloning the target gene on TRV2. We then inserted Thaumatin on the TRV2 vector and co-injected it with the TRV1 vector into tomatoes. As the virus replicates autonomously within the tomato cells, it can utilize intracellular substances to express Thaumatin.

At the same time, we inhibited the RNA interference system of tomato plants themselves by introducing silencing repressor P19 to ensure that the viral vectors could remain in tomato for a period of time. In addition, in order to monitor the effectiveness of the viral vector, we introduced mCherry red fluorescent protein partial sequence as a control.

Fig 4. The Thaumatin expression pathway constructed in the TRV system.
(A) The mechanism of the introduction of the target fragment into tomato cell through the TRV vector.
(B) The expression pathway of Thaumatin.
(C) The expression pathway of P19.
(D) The expression pathway of the partial sequence of mCherry.

Pathway optimization

We verified that Thaumatin is correctly expressed in tomato. In order to make the production of Thaumatin sustainable, we have changed the methods of production. Transgenic technology enables the introduction of exogenous gene sequences into the host genome, resulting in heritable changes in biological traits. Not only has it been used for a long time as a traditional breeding method, but it has also been instrumental in the production of products in bioreactors.

Transgenic technology is based on a sequence of T-DNA on the plasmid of Agrobacterium. After Agrobacterium invades plant cells through wounds, it inserts the T-DNA into the genome of the target plant.

Agrobacterium Ti plasmid is a double-stranded covalently closed circular DNA molecule. It contains four functional regions, which are T-DNA region, Vir region, Con region and Ori region. The T-DNA region is the fragment that can be transferred and integrated into the plant genome; the Vir region encodes proteins that are involved in the processing and transfer of T-DNA; the Con region is related to the binding and transfer of the plasmid; and the Ori region is the plasmid replication initiation site.

When the plant is wounded, the damage cells will secrete a large number of phenolic compounds. These phenolics can induce the expression of Vir genes in Agrobacterium. Through chemotaxis, Agrobacterium recognizes and attaches to the wounded area of the plant. The expression product of the Vir gene cuts off a single strand of T-DNA from the Ti plasmid to form a single-stranded T-DNA. Subsequently, the single-stranded T-DNA binds to the expression products of the Vir gene and the Con gene to form a complex. The complex transfers the T-DNA to the plant cell through contact between Agrobacterium and the plant cell. Upon entering the plant cell, the T-DNA is able to randomly integrate into the plant chromosome. This process can be single-copy or multi-copy, but is usually preferred near the coding or regulatory regions of a gene.

Fig 5. The mechanism of transgenic technology with Agrobacterium.

In the conventional transgenic pathway, synthesis of Thaumatin and Brazzein initiates expression under the CaMV 35S promoter. However, the CaMV 35S promoter lacks stability. On the one hand, a large number of studies have shown that although the CaMV 35S promoter is highly expressed, it affects the expression of neighboring genes, leading to disease in plants. On the other hand, the activity of CaMV 35S promoter varies in different tissues and cells of plants, making it difficult to ensure the expression level.

In order to ensure the stability of sweet protein expression in transgenic tomato, we need to optimize the promoter. Therefore, we chose the tomato fruit-specific promoter E8 instead of the 35S promoter. This tissue-specific promoter can not only accumulate the expression products of target genes in certain organs or tissues and increase regional expression, but also avoid unnecessary waste of plant nutrition.

The E8 promoter has a highly specific expression pattern in tomato fruit. It is mainly activated at specific stages of tomato fruit development, driving preferential expression of downstream genes in fruit tissues. The E8 promoter is significantly more active in fruit compared to other tissues such as roots, stems and leaves. It can be utilized to achieve precise regulation of specific genes in fruits. By linking the target gene to the E8 promoter, the target gene can be expressed only in tomato fruits, avoiding unwanted effects in other tissues. This is important for improving the quality and characteristics of tomato fruits.

Fig 6. Functions of fruit-specific promoter E8.

During fruit ripening, the E8 promoter can initiate the expression of a series of genes that are involved in physiological processes such as fruit color change, texture softening, and flavor substance synthesis. These changes are the result of the mutual regulation of the E8 promoter and ethylene.

There are at least two major regions in the E8 promoter sequence that contribute to its transcriptional regulation. One is the upstream region from -2181 to -1088, which contains ethylene-responsive transcriptional elements. When ethylene concentration is elevated, the expression of the E8 promoter and ethylene synthesis genes increases simultaneously, forming a positive feedback regulatory mechanism that further promotes fruit ripening. And in the absence of ethylene synthesis, another region comes into play. The sequence from -409 to -263 of the transcription start site is sufficient for ripening-specific transcription.

Storage location

Vacuole, as compartment that occupy most of the space in tomato fruit cells, has the following advantages in storing Thaumatin:

pH environmental aspects:
The liquid environment ph inside the vacuoles is acidic, adapting to the acid stability characteristics of the soluble protein Thaumatin.

Aspects of the contents:
Plants form special vacuoles containing large amounts of specific substances in specific tissues. Plant vacuoles are mainly divided into two distinct chambers: storage chamer and lysis chamber. The typical storage compartment is the protein storage vacuole (PSV) found in seeds, and the typical lysis compartment is the lysing vacuole (LV) found in leaf pulp cells, whereas the PSV contains far fewer protein hydrolyzing enzymes than the LV.

In terms of tissue relationships, tomato fruits and seeds are storage tissues that are more similar in their vacuole properties. As a result, the vesicles within the fruit contain less hydrolytic enzymes and are therefore more suitable for Thaumatin storage.

Fruit development:
Tomato, as a berry, undergoes complex processes such as the transformation of chloroplasts to colored bodies, softening of the cell wall, accumulation of pigments, and changes in hormone levels in the cells during fruit ripening. At this time, the cytoplasmic fluid environment is complex, which is not conducive to the stable existence of Thaumatin. On the contrary, the environment within the vacuole is in a relatively stable state, therefore, the vacuole compartment prevents the effect of the huge environmental changes on Thaumatin during fruit ripening.

We attached the sequence of glycosporine-N-terminal prepeptide SPS-NTPP (GenBank accession number: EF638829). It is a vacuolar transport signaling molecule that precisely and efficiently escorts thaumatin to the vesicles for storage, and after targeting is complete SPS-NTPP is able to self-decompose without any effect, dramatically increasing thaumatin content in tomato. Therefore, we constructed the pGD-SPS-NTPP-thaumatin-EGFP plasmid and transformed it into Agrobacterium GV3101 for transient infestation of Nicotiana benthamiana, thus verifing the targeting function of SPS-NTPP in tobacco leaves.

Fig 7. Schematic diagram of the localization of SPS-NTPP

Plasmid construction

In prokaryotes

We chose one of the most common strains E. coli BL21(DE3) for fermentation production. The target sequence we designed was controlled by the T7 promoter on the plasmid pET-28a(+) to obtain our expression vector. Subsequently, we transformed the vector into E. coli BL21(DE3) and picked the colonies for amplification on the medium containing kanamycin.

Fig 8. Construction of prokaryotic recombinant vector.

In plants

We constructed the TRV2-35S-Thaumatin plasmid using the virus TRV (Tobacco rattle virus) and infested tomato plants. We used this transient infestation to verify the viability of tomato chassis.

Fig 9. Construction of TRV2-35S-Thaumatin vector.

Next, we constructed CaMV 35S-Thaumatin and E8-Thaumatin plasmids using the binary vectors pBWA(V)HS and pCAMBIA1301 as backbones for tomato transgenic expression respectively.

Fig 10. Construction of pBWA(V)HS-35S-Thaumatin and pCAMBIA1301-E8-Thaumatin vectors.

To validate that the incorporation of SPS-NTPP can direct Thaumatin to be localized in the vacuole, we initially aimed to reproduce the localization effect of this targeting peptide in our laboratory, which was a guarantee indicating that we could proceed to the next stage of experiments. The most expedient approach was to link SPS-NTPP with Thaumatin and attach an EGFP protein at the rear end. The fluorescence emitted by it could thereby indicate the position of Thaumatin. Hence, we constructed the pGD_SPS-NTPP-Thaumatin-EGFP plasmid.

Fig 11. Construction of pGD_SPS-NTPP-Thaumatin-EGFP vectors.

Characterization

Synthetic validation

Ⅰ. Expression prokaryotic
We extracted bacterial proteins for Western Blot to detect whether Thaumatin is expressed or not. We found that the expression of Thaumatin in E. coli was not satisfactory after induction by IPTG (Fig 12). During the experiment, we found that E. coli was prone to forming inclusion bodies which made it extremely difficult to extract the protein, so we improved the experimental method. We disrupted the cells, centrifuged them to get the supernatant for Western Blot, but the results still showed that the expression of Thaumatin was extremely low. This indicates that E. coli is not suitable as a chassis for Thaumatin expression and we need to change the expression system.

Fig 12. SDS-PAGE and WB analysis for Thaumatin cloned in pET28a. M1/M2: marker;
PC1: BSA (1 μg); PC2: BSA (2 μg); NC: Cell lysate without induction;
Lane 1: Cell lysate with induction for 16 h at 15℃; Lane 2: Cell lysate with induction for 4 h at 37℃;
Lane NC1: Supernatant of cell lysate without induction; Lane 3: Supernatant of cell lysate with induction for 16 h at 15℃;
Lane 4: Supernatant of cell lysate with induction for 4 h at 37℃; Lane NC2: Pellet of cell lysate without induction;
Lane 5: Pellet of cell lysate with induction for 16 h at 15℃; Lane 6: Pellet of cell lysate with induction for 4 h at 37℃.

Ⅱ. Validation of tomato chassis
(1)Agarose gel electrophoresis:
The colonies were amplified by PCR with specific primers and the products were obtained and then subjected to agarose gel electrophoresis. From the results, it can be seen that the band of Thaumatin (719 bp) has a band appearing near 700 bp (Fig 13). This can verify that our transformed Agrobacterium has carried on the target sequence. This is an important milestone and lays the foundation for further experiments.

Fig 13. PCR identification of Agrobacterium after transformation

(2)RNA expression test:
After ensuring that the plasmid was successfully transferred into Agrobacterium GV3101, we amplified Agrobacterium into culture and assembled the virus by inducing it with MMA. Next, we injected the virus into the leaves of four-leaf-old micro-TOM. After the plants grew up, the leaves were collected for RNA extraction and RT-PCR experiments were performed to verify the transcription of Thaumatin gene in the leaves. According to the results, it can be seen that the RNA band of Thaumatin (719 bp) has a band near 700 bp, which indicates that Thaumatin has begun to be expressed in tomato plants, and we can proceed to the next step of verification (Fig 14).

Fig 14. Plot of RNA extracted from leaves for RT-PCR results.

(3)Protein expression:
We collected leaves, flowers and fruits of TRV virus-infected tomato plants and extracted proteins for WB assay. According to the results, it was clear that tomato was able to express Thaumatin. Based on the immunoenzymatic ELISA for Thaumatin, we concluded that the average level of transient expression of Thaumatin was 11.2873 mg/L in the virus-infested tomato experiment (Fig 15).

Fig 15. (A)Western Blot results for Thaumatin after TRV infection to leaves of tomato.
(B)Western Blot results for Thaumatin after TRV infection to fruits of tomato.
(C) Detection of Thaumatin in tomato fruits infected with TRV.

Ⅲ. Whole-plant expression
(1)Agarose gel electrophoresis:
To confirm the plasmid was transformed into Agrobacterium, a fragement on hygromycin B resistance gene HygR was amplified by PCR with specific primers and the products were obtained and then subjected to agarose gel electrophoresis. From the results, it can be seen that the amplification result of the fragment (719 bp) has a band appearing near 700bp (Fig 16). This can be verified that our transformed agrobacterium has carried the target sequence.

Fig 16. Successful transformation of Agrobacterium.
(C) Detection of Thaumatin in tomato fruits infected with TRV.

(2)DNA test:
We chose positive bacteria to infect the callus tissue, and when the callus tissue grew to a certain extent, it was inoculated and cultivated in rooting medium (Fig 17), and when the plant grew leaves, we picked the leaves to test whether the plasmid was inseted into the genome of tomato. The results of the analysis showed that HygR DNA could be detected (Fig 18).

Fig 17. Callus tissues cultivation after infection

Fig 18. PCR amplification of the Thaumatin DNA in tomato leaves

(3)Protein expression:
We collected leaf, flower and fruit samples, extracted proteins for Western Blot assay and successfully obtained positive results around 28 kD, indicating successful expression of Thaumatin (28.36 kD) in transgenic micro-TOM (Fig 19A,B,C). This is the first milestone in our experimental process, indicating that transgenic tomatoes with stable genetic ability to produce sweet proteins have been successfully bred. Based on the ELISA for Thaumatin, we concluded that the average level of transient expression of Thaumatin in the virus-infested tomato experiment was 11.1598 mg/L (Fig 19D).

Fig 19. Western Blot results of the (A)leaves, (B)flowers, and (C)fruits of transgenic tomato
plants expressing Thaumatin under the 35S promoter.(D)Thuamatin expression of tomato fruit controlled by 35S promoter.

Ⅳ. Specific expression
(1)Transformation success:
We used specific primers for PCR and proved the success of our expression vector into Agrobacterium GV3101 by agarose gel electrophoresis (Fig 20).

Fig 20. Agarose gel electrophoresis is used to detect plasmid transformation.

(2)Expression at DNA level:
After the plants grew leaves, we picked the leaves to test whether our genes were present in the genome of tomato. The results of the analysis showed that all of Thaumatin's DNA could be detected (Fig 21).

Fig 21. Detect plasmid DNA in transgenic tomatoes.

(3)Expression at protein level:
We harvested fruit samples and extracted proteins for WB assay. Eventually, we only succeeded in detecting the presence of a positive band around 28 kD in the fruit, but not in the leaves and flowers (Fig 22). This result suggests that Thaumatin (28.36 kD) was successfully expressed in our transgenic tomato under the initiation of the E8 promoter.

Fig 22 Protein expression of Thaumatin.
(A) Protein was examined in transgenic tomato leaf with the E8 promoter.
(B) Protein was examined in transgenic tomato flower with the E8 promoter.
(C) Protein was examined in transgenic tomato fruits with the E8 promoter. 

We analyzed the efficacy of the E8 promoter to express proteins. We determined the concentration of Thaumatin expressed with the CaMV 35S promoter and the E8 promoter in transgenic tomatoes by ELISA. By comparison, we obtained that the average level of Thaumatin expressed under the induction of the constitutive promoter 35S was 11.1598 mg/L; while the expression level using the fruit ripening-specific promoter was 11.0591 mg/L (Fig 23). There was no statistically significant difference, indicating that the expression level of the fruit-specific promoter E8 had reached the expression level of the strong promoter CaMV 35S. Compared with 35S, the E8 promoter not only ensures fruit-specific expression and avoids burdening other parts of the tomato plant, but also ensures the high expression level of Thaumatin.

Fig 23. Comparison of the amount of Thaumatin expressed by the 35S promoter and the E8 promoter

@@ Line 2: / Line 2: @@
 __NOTOC__
 <partinfo>BBa_K5160003 short</partinfo>
+==Overview==
+In order to provide people with a safe sweetener, our project hopes to propose a method for the efficient production of Thaumatin. Thaumatin comes from a tropical fruit in Africa and has been certified by the FDA and GRAS as a safe and reliable sugar substitute. Thaumatin binds to receptors on the human tongue to induce a sweet taste. This process produces almost no calories. At the same time, It can be fully digested by the human body and produce no side effects. This shows that Thaumatin is a very safe and reliable sweetener, which makes it an excellent choice for sugar reduction and control. About the engineering cycle and iteration of Thaumatin. Please go to the engineering section to find out more about the engineering cycle and the iteration of Thaumatin production.
+<span class='h3bb'>Sequence and Features</span>
+<partinfo>BBa_K5160003 SequenceAndFeatures</partinfo>
+<br />
+==Usage and biology==
+Thaumatin is derived from the aril of the tropical plant Thaumatococcus daniellii (Benth). Bamboo yam contains two types of Thaumatin: Thaumatin I and Thaumatin II. One of them, Thaumatin II, has a higher sweetness. It consists of 207 amino acids, and the amino acids are coiled and folded to form eight disulfide bonds. As a result, the protein structure of Thaumatin is stable, thermally and acid-stable.<br />
+<br />
+Thaumatin belongs to the family of five pathogenesis-related proteins (PR5), which are characterized by significant antifungal as well as phytopathogenic activity. Thus, in bamboo yam, the main function of Thaumatin is to protect seeds and fruits against abiotic stresses. After discovering the sweet flavor characteristics of Thaumatin, people have developed it and used it in various foods.<br />
+<br />
+Thaumatin binds to sweetness receptors T1R2 and T1R3 on the human tongue, activating G-protein-coupled receptors on the membrane surface, generating an electrical signal that is transmitted to the cerebral cortex, causing sweetness perception. As a protein, Thaumatin consists of common amino acids and can be fully digested and absorbed by the body without generating calories.<br />
+<br />
+<center><html><img src="https://static.igem.wiki/teams/5160/project/engineering/1.png" width="50%" height="50%"  /></html></center>
+<center><b>Fig 1. Origin and protein structure of Thaumatin.</b></center>
+<br />
+<br />
+==Design==
+===Sequence identification===
+We found the original record of heterologous expression of Thaumatin from the literature. However, the recorded fragment was incomplete, so we blasted the incomplete sequence in NCBI to obtain the complete cds sequence. Finally, we obtained the complete sequence of Thaumatin II on the whole genome.<br />
+<center><html><img src="https://static.igem.wiki/teams/5160/project/engineering/2.png" width="70%" height="65%"  /></html></center>
+<center><b>Fig 2. Nucleotide BLAST results on NBCI.</b></center>
+<br />
+===Chassis selection===
+Because of the complex structure of Thaumatin, we hoped to find a suitable chassis to express it efficiently.<br />
+<br />
+The successful expression of the protein is related to its size, but it mainly depends on whether the protein expressed in the chassis can be properly transcribed, translated and folded.<br />
+<br />
+Firstly, we tried to express Thaumatin and Brazzein in E. coli by constructing expression vectors and transformed them into E. coli, then induced the expression with IPTG and tested the protein level. Both Thaumatin and Brazzein have the special structure of disulfide bond, and the prokaryotic expression system has natural defects, which is not able to fold proteins correctly to form a complex structure, so the protein expression results were not satisfactory. <br />
+<center><html><img src="https://static.igem.wiki/teams/5160/project/engineering/3.png" width="50%" height="50%"  /></html></center>
+<center><b>Fig 3. The expression of Thaumatin in the prokaryotic system.</b></center>
+<br />
+Considering the practical application of sugar substitutes as a food additive, and the fact that fermentation production requires the complex process of protein purification, which may introduce safety concerns, we aim to simplify the Thaumatin production process by eliminating the need for purification steps. Thus, we have turned our focus to a fruit commonly consumed in daily life—tomatoes.<br />
+<br />
+===Pathway design===
+To validate the feasibility of the tomato platform, we need a quick testing method. Therefore, we aim to achieve this goal through transient expression. Many studies have shown that transient infection can be accomplished by Agrobacterium dipping or by viral vector leaf injection. Comparing the two methods, we found that Agrobacterium dipping struggles to maintain traits. A more critical issue is that it is difficult for it to complete infection in tomato fruits. In contrast, viral expression vectors can maintain traits for a longer period, which is advantageous for testing in tomato fruits. Therefore, we chose to use viral expression vectors for this step of validation.<br />
+<br />
+TRV is a double-stranded RNA virus with a genome consisting of RNA1 and RNA2. With the reverse transcriptase, the TRV genome is reverse transcribed into cDNA in vitro. Specific modifications and cloning of the cDNA transform it into a non-toxic vector that retains both self-replication ability and the ability to express foreign genes, known as TRV1 and TRV2. The TRV1 vector usually contains genes encoding RNA-dependent RNA polymerase (RdRp), movement protein (MP), and a 16 kDa protein. It serves as an auxiliary viral vector to assist in the replication and spread of the virus within the plant. TRV2 contains the gene encoding the viral coat protein, and an artificially inserted MCS sequence is used for cloning the target gene on TRV2. We then inserted Thaumatin on the TRV2 vector and co-injected it with the TRV1 vector into tomatoes. As the virus replicates autonomously within the tomato cells, it can utilize intracellular substances to express Thaumatin.<br />
+<br />
+At the same time, we inhibited the RNA interference system of tomato plants themselves by introducing silencing repressor P19 to ensure that the viral vectors could remain in tomato for a period of time. In addition, in order to monitor the effectiveness of the viral vector, we introduced mCherry red fluorescent protein partial sequence as a control. <br />
+<br />
+<center><html><img src="https://static.igem.wiki/teams/5160/project/engineering/4.png" width="70%" height="50%"  /></html></center>
+<center><b>Fig 4. The Thaumatin expression pathway constructed in the TRV system. <br />(A) The mechanism of the introduction of the target fragment into tomato cell through the TRV vector.<br /> (B) The expression pathway of Thaumatin. <br />(C) The expression pathway of P19.<br /> (D) The expression pathway of the partial sequence of mCherry.</b></center>
+<br />
+<br />
+===Pathway optimization===
+We verified that Thaumatin is correctly expressed in tomato. In order to make the production of Thaumatin sustainable, we have changed the methods of production. Transgenic technology enables the introduction of exogenous gene sequences into the host genome, resulting in heritable changes in biological traits. Not only has it been used for a long time as a traditional breeding method, but it has also been instrumental in the production of products in bioreactors. <br />
+<br />
+Transgenic technology is based on a sequence of T-DNA on the plasmid of Agrobacterium. After Agrobacterium invades plant cells through wounds, it inserts the T-DNA into the genome of the target plant.<br />
+<br />
+Agrobacterium Ti plasmid is a double-stranded covalently closed circular DNA molecule. It contains four functional regions, which are T-DNA region, Vir region, Con region and Ori region. The T-DNA region is the fragment that can be transferred and integrated into the plant genome; the Vir region encodes proteins that are involved in the processing and transfer of T-DNA; the Con region is related to the binding and transfer of the plasmid; and the Ori region is the plasmid replication initiation site.<br />
+<br />
+When the plant is wounded, the damage cells will secrete a large number of phenolic compounds. These phenolics can induce the expression of Vir genes in Agrobacterium. Through chemotaxis, Agrobacterium recognizes and attaches to the wounded area of the plant. The expression product of the Vir gene cuts off a single strand of T-DNA from the Ti plasmid to form a single-stranded T-DNA. Subsequently, the single-stranded T-DNA binds to the expression products of the Vir gene and the Con gene to form a complex. The complex transfers the T-DNA to the plant cell through contact between Agrobacterium and the plant cell. Upon entering the plant cell, the T-DNA is able to randomly integrate into the plant chromosome. This process can be single-copy or multi-copy, but is usually preferred near the coding or regulatory regions of a gene.<br />
+<br />
+<center><html><img src="https://static.igem.wiki/teams/5160/project/engineering/5.png" width="55%" height="50%"  /></html></center>
+<center><b>Fig 5. The mechanism of transgenic technology with Agrobacterium.</b></center>
+<br />
+In the conventional transgenic pathway, synthesis of Thaumatin and Brazzein initiates expression under the CaMV 35S promoter. However, the CaMV 35S promoter lacks stability. On the one hand, a large number of studies have shown that although the CaMV 35S promoter is highly expressed, it affects the expression of neighboring genes, leading to disease in plants. On the other hand, the activity of CaMV 35S promoter varies in different tissues and cells of plants, making it difficult to ensure the expression level.<br />
+<br />
+In order to ensure the stability of sweet protein expression in transgenic tomato, we need to optimize the promoter. Therefore, we chose the tomato fruit-specific promoter E8 instead of the 35S promoter. This tissue-specific promoter can not only accumulate the expression products of target genes in certain organs or tissues and increase regional expression, but also avoid unnecessary waste of plant nutrition.<br />
+<br />
+The E8 promoter has a highly specific expression pattern in tomato fruit. It is mainly activated at specific stages of tomato fruit development, driving preferential expression of downstream genes in fruit tissues. The E8 promoter is significantly more active in fruit compared to other tissues such as roots, stems and leaves. It can be utilized to achieve precise regulation of specific genes in fruits. By linking the target gene to the E8 promoter, the target gene can be expressed only in tomato fruits, avoiding unwanted effects in other tissues. This is important for improving the quality and characteristics of tomato fruits.<br />
+<br />
+<center><html><img src="https://static.igem.wiki/teams/5160/project/engineering/6.jpg" width="50%" height="50%"  /></html></center>
+<center><b>Fig 6. Functions of fruit-specific promoter E8.</b></center>
+<br />
+During fruit ripening, the E8 promoter can initiate the expression of a series of genes that are involved in physiological processes such as fruit color change, texture softening, and flavor substance synthesis. These changes are the result of the mutual regulation of the E8 promoter and ethylene.<br />
+<br />
+There are at least two major regions in the E8 promoter sequence that contribute to its transcriptional regulation. One is the upstream region from -2181 to -1088, which contains ethylene-responsive transcriptional elements. When ethylene concentration is elevated, the expression of the E8 promoter and ethylene synthesis genes increases simultaneously, forming a positive feedback regulatory mechanism that further promotes fruit ripening. And in the absence of ethylene synthesis, another region comes into play. The sequence from -409 to -263 of the transcription start site is sufficient for ripening-specific transcription.<br />
+<br />
+===Storage location===
+Vacuole, as compartment that occupy most of the space in tomato fruit cells, has the following advantages in storing Thaumatin:<br />
+<br />
+<b>pH environmental aspects:</b><br />
+The liquid environment ph inside the vacuoles is acidic, adapting to the acid stability characteristics of the soluble protein Thaumatin.<br />
+<br />
+<b>Aspects of the contents:</b><br />
+Plants form special vacuoles containing large amounts of specific substances in specific tissues. Plant vacuoles are mainly divided into two distinct chambers: storage chamer and lysis chamber. The typical storage compartment is the protein storage vacuole (PSV) found in seeds, and the typical lysis compartment is the lysing vacuole (LV) found in leaf pulp cells, whereas the PSV contains far fewer protein hydrolyzing enzymes than the LV.<br />
+<br />
+In terms of tissue relationships, tomato fruits and seeds are storage tissues that are more similar in their vacuole properties. As a result, the vesicles within the fruit contain less hydrolytic enzymes and are therefore more suitable for Thaumatin storage.<br />
+<br />
+<b>Fruit development:</b><br />
+Tomato, as a berry, undergoes complex processes such as the transformation of chloroplasts to colored bodies, softening of the cell wall, accumulation of pigments, and changes in hormone levels in the cells during fruit ripening. At this time, the cytoplasmic fluid environment is complex, which is not conducive to the stable existence of Thaumatin. On the contrary, the environment within the vacuole is in a relatively stable state, therefore, the vacuole compartment prevents the effect of the huge environmental changes on Thaumatin during fruit ripening.<br />
+<br />
+We attached the sequence of glycosporine-N-terminal prepeptide SPS-NTPP (GenBank accession number: EF638829). It is a vacuolar transport signaling molecule that precisely and efficiently escorts thaumatin to the vesicles for storage, and after targeting is complete SPS-NTPP is able to self-decompose without any effect, dramatically increasing thaumatin content in tomato. Therefore, we constructed the pGD-SPS-NTPP-thaumatin-EGFP plasmid and transformed it into Agrobacterium GV3101 for transient infestation of Nicotiana benthamiana, thus verifing the targeting function of SPS-NTPP in tobacco leaves.<br />
+<br />
+<center><html><img src="https://static.igem.wiki/teams/5160/project/engineering/7.png" width="50%" height="50%"  /></html></center>
+<center><b>Fig 7. Schematic diagram of the localization of SPS-NTPP</b></center>
+<br />
+<br />
+==Plasmid construction==
+===In prokaryotes===
+We chose one of the most common strains E. coli BL21(DE3) for fermentation production. The target sequence we designed was controlled by the T7 promoter on the plasmid pET-28a(+) to obtain our expression vector. Subsequently, we transformed the vector into E. coli BL21(DE3) and picked the colonies for amplification on the medium containing kanamycin.<br />
+<br />
+<center><html><img src="https://static.igem.wiki/teams/5160/wet-lab/result/e-coli-t.png" width="40%" height="50%"  /></html></center>
+<center><b>Fig 8. Construction of prokaryotic recombinant vector.</b></center>
+<br />
+===In plants===
+We constructed the TRV2-35S-Thaumatin plasmid using the virus TRV (Tobacco rattle virus) and infested tomato plants. We used this transient infestation to verify the viability of tomato chassis. <br />
+<br />
+<center><html><img src="https://static.igem.wiki/teams/5160/wet-lab/result/pl-trv2-thau.webp" width="40%" height="50%"  /></html></center>
+<center><b>Fig 9. Construction of TRV2-35S-Thaumatin vector.</b></center>
+<br />
+Next, we constructed CaMV 35S-Thaumatin and E8-Thaumatin plasmids using the binary vectors pBWA(V)HS and pCAMBIA1301 as backbones for tomato transgenic expression respectively.<br />
+<center><html><img src="https://static.igem.wiki/teams/5160/project/engineering/10.png" width="75%" height="50%"  /></html></center>
+<center><b>Fig 10. Construction of pBWA(V)HS-35S-Thaumatin and pCAMBIA1301-E8-Thaumatin vectors.</b></center>
+<br />
+To validate that the incorporation of SPS-NTPP can direct Thaumatin to be localized in the vacuole, we initially aimed to reproduce the localization effect of this targeting peptide in our laboratory, which was a guarantee indicating that we could proceed to the next stage of experiments. The most expedient approach was to link SPS-NTPP with Thaumatin and attach an EGFP protein at the rear end. The fluorescence emitted by it could thereby indicate the position of Thaumatin. Hence, we constructed the pGD_SPS-NTPP-Thaumatin-EGFP plasmid. <br />
+<br />
+<center><html><img src="https://static.igem.wiki/teams/5160/wet-lab/result/pgd-sps-ntpp.png" width="45%" height="50%"  /></html></center>
+<center><b>Fig 11. Construction of pGD_SPS-NTPP-Thaumatin-EGFP vectors.</b></center>
+<br />
+==Characterization==
+===Synthetic validation===
+<b> Ⅰ. Expression prokaryotic</b><br />
+We extracted bacterial proteins for Western Blot to detect whether Thaumatin is expressed or not. We found that the expression of Thaumatin in E. coli was not satisfactory after induction by IPTG (Fig 12). During the experiment, we found that E. coli was prone to forming inclusion bodies which made it extremely difficult to extract the protein, so we improved the experimental method. We disrupted the cells, centrifuged them to get the supernatant for Western Blot, but the results still showed that the expression of Thaumatin was extremely low. This indicates that E. coli is not suitable as a chassis for Thaumatin expression and we need to change the expression system.<br />
+<center><html><img src="https://static.igem.wiki/teams/5160/project/engineering/12.png" width="60%" height="45%"  /></html></center>
+<center><b>Fig 12. SDS-PAGE and WB analysis for Thaumatin cloned in pET28a. M1/M2: marker;<br /> PC1: BSA (1 μg); PC2: BSA (2 μg); NC: Cell lysate without induction;<br /> Lane 1: Cell lysate with induction for 16 h at 15℃; Lane 2: Cell lysate with induction for 4 h at 37℃;<br /> Lane NC1: Supernatant of cell lysate without induction; Lane 3: Supernatant of cell lysate with induction for 16 h at 15℃;<br /> Lane 4: Supernatant of cell lysate with induction for 4 h at 37℃; Lane NC2: Pellet of cell lysate without induction; <br />Lane 5: Pellet of cell lysate with induction for 16 h at 15℃; Lane 6: Pellet of cell lysate with induction for 4 h at 37℃. </b></center>
+<br />
+<b> Ⅱ. Validation of tomato chassis</b><br />
+(1)Agarose gel electrophoresis:<br />
+The colonies were amplified by PCR with specific primers and the products were obtained and then subjected to agarose gel electrophoresis. From the results, it can be seen that the band of Thaumatin (719 bp) has a band appearing near 700 bp (Fig 13). This can verify that our transformed Agrobacterium has carried on the target sequence. This is an important milestone and lays the foundation for further experiments.<br />
+<center><html><img src="https://static.igem.wiki/teams/5160/wet-lab/outline/image-3.png" width="66%" height="50%"  /></html></center>
+<center><b>Fig 13.  PCR identification of Agrobacterium after transformation</b></center>
+<br />
+(2)RNA expression test:<br />
+After ensuring that the plasmid was successfully transferred into Agrobacterium GV3101, we amplified Agrobacterium into culture and assembled the virus by inducing it with MMA. Next, we injected the virus into the leaves of four-leaf-old micro-TOM. After the plants grew up, the leaves were collected for RNA extraction and RT-PCR experiments were performed to verify the transcription of Thaumatin gene in the leaves. According to the results, it can be seen that the RNA band of Thaumatin (719 bp) has a band near 700 bp, which indicates that Thaumatin has begun to be expressed in tomato plants, and we can proceed to the next step of verification (Fig 14).<br />
+<center><html><img src="https://static.igem.wiki/teams/5160/wet-lab/outline/trv-leaf-thau.png" width="60%" height="50%"  /></html></center>
+<center><b>Fig 14. Plot of RNA extracted from leaves for RT-PCR results.</b></center>
+<br />
+(3)Protein expression:<br />
+We collected leaves, flowers and fruits of TRV virus-infected tomato plants and extracted proteins for WB assay. According to the results, it was clear that tomato was able to express Thaumatin. Based on the immunoenzymatic ELISA for Thaumatin, we concluded that the average level of transient expression of Thaumatin was 11.2873 mg/L in the virus-infested tomato experiment (Fig 15).<br />
+<center><html><img src="https://static.igem.wiki/teams/5160/project/engineering/15.png" width="60%" height="50%"  /></html></center>
+<center><b>Fig 15. (A)Western Blot results for Thaumatin after TRV infection to leaves of tomato. <br /> (B)Western Blot results for Thaumatin after TRV infection to fruits of tomato.<br />  (C) Detection of Thaumatin in tomato fruits infected with TRV.</b></center>
+<br />
+<b> Ⅲ. Whole-plant expression</b><br />
+(1)Agarose gel electrophoresis:<br />
+To confirm the plasmid was transformed into Agrobacterium, a fragement on hygromycin B resistance gene HygR was amplified by PCR with specific primers and the products were obtained and then subjected to agarose gel electrophoresis. From the results, it can be seen that the amplification result of the fragment (719 bp) has a band appearing near 700bp (Fig 16). This can be verified that our transformed agrobacterium has carried the target sequence.<br />
+<center><html><img src="https://static.igem.wiki/teams/5160/project/engineering/16.png" width="40%" height="50%"  /></html></center>
+<center><b>Fig 16. Successful transformation of Agrobacterium.<br />  (C) Detection of Thaumatin in tomato fruits infected with TRV.</b></center>
+<br />
+(2)DNA test:<br />
+We chose positive bacteria to infect the callus tissue, and when the callus tissue grew to a certain extent, it was inoculated and cultivated in rooting medium (Fig 17), and when the plant grew leaves, we picked the leaves to test whether the plasmid was inseted into the genome of tomato. The results of the analysis showed that HygR DNA could be detected (Fig 18).<br />
+<center><html><img src="https://static.igem.wiki/teams/5160/project/engineering/17.png" width="50%" height="50%"  /></html></center>
+<center><b>Fig 17. Callus tissues cultivation after infection</b></center>
+<br />
+<br />
+<center><html><img src="https://static.igem.wiki/teams/5160/project/engineering/18.png" width="55%" height="50%"  /></html></center>
+<center><b>Fig 18. PCR amplification of the Thaumatin DNA in tomato leaves</b></center>
+<br />
+(3)Protein expression:<br />
+We collected leaf, flower and fruit samples, extracted proteins for Western Blot assay and successfully obtained positive results around 28 kD, indicating successful expression of Thaumatin (28.36 kD) in transgenic micro-TOM (Fig 19A,B,C). This is the first milestone in our experimental process, indicating that transgenic tomatoes with stable genetic ability to produce sweet proteins have been successfully bred.
+Based on the ELISA for Thaumatin, we concluded that the average level of transient expression of Thaumatin in the virus-infested tomato experiment was 11.1598 mg/L (Fig 19D).<br />
+<center><html><img src="https://static.igem.wiki/teams/5160/project/engineering/19.png" width="70%" height="50%"  /></html></center>
+<center><b>Fig 19. Western Blot results of the (A)leaves, (B)flowers, and (C)fruits of transgenic tomato<br /> plants expressing Thaumatin under the 35S promoter.(D)Thuamatin expression of tomato fruit controlled by 35S promoter.</b></center>
+<br />
+<b> Ⅳ. Specific expression<b/><br />
+(1)Transformation success:<br />
+We used specific primers for PCR and proved the success of our expression vector into Agrobacterium GV3101 by agarose gel electrophoresis (Fig 20). <br />
+<center><html><img src="https://static.igem.wiki/teams/5160/wet-lab/result/e8-bra-eb-thau-a-gv3101.webp" width="50%" height="50%"  /></html></center>
+<center><b>Fig 20. Agarose gel electrophoresis is used to detect plasmid transformation.</b></center>
+<br />
+(2)Expression at DNA level:<br />
+After the plants grew leaves, we picked the leaves to test whether our genes were present in the genome of tomato. The results of the analysis showed that all of Thaumatin's DNA could be detected (Fig 21).<br />
+<center><html><img src="https://static.igem.wiki/teams/5160/wet-lab/result/e8-thaudna2.png" width="50%" height="50%"  /></html></center>
+<center><b>Fig 21. Detect plasmid DNA in transgenic tomatoes.</b></center>
+<br />
+(3)Expression at protein level:<br />
+We harvested fruit samples and extracted proteins for WB assay. Eventually, we only succeeded in detecting the presence of a positive band around 28 kD in the fruit, but not in the leaves and flowers (Fig 22). This result suggests that Thaumatin (28.36 kD) was successfully expressed in our transgenic tomato under the initiation of the E8 promoter.<br />
+<center><html><img src="https://static.igem.wiki/teams/5160/project/engineering/22.png" width="75%" height="50%"  /></html></center>
+<center><b>Fig 22 Protein expression of Thaumatin. <br />(A) Protein was examined in transgenic tomato leaf with the E8 promoter.<br /> (B) Protein was examined in transgenic tomato flower with the E8 promoter.<br /> (C) Protein was examined in transgenic tomato fruits with the E8 promoter. </b></center>
+<br />
+We analyzed the efficacy of the E8 promoter to express proteins. We determined the concentration of Thaumatin expressed with the CaMV 35S promoter and the E8 promoter in transgenic tomatoes by ELISA. By comparison, we obtained that the average level of Thaumatin expressed under the induction of the constitutive promoter 35S was 11.1598 mg/L; while the expression level using the fruit ripening-specific promoter was 11.0591 mg/L (Fig 23). There was no statistically significant difference, indicating that the expression level of the fruit-specific promoter E8 had reached the expression level of the strong promoter CaMV 35S. Compared with 35S, the E8 promoter not only ensures fruit-specific expression and avoids burdening other parts of the tomato plant, but also ensures the high expression level of Thaumatin.<br />
+<center><html><img src="https://static.igem.wiki/teams/5160/wet-lab/result/cthuamatin-of-fruit-35s.png" width="40%" height="50%"  /></html><html><img src="https://static.igem.wiki/teams/5160/wet-lab/result/cthuamatin-of-fruit-e8.png" width="40%" height="50%"  /></html><html><img src="https://static.igem.wiki/teams/5160/wet-lab/result/cthuamatin-of-fruit-all.png" width="40%" height="50%"  /></html></center>
+<center><b>Fig 23. Comparison of the amount of Thaumatin expressed by the 35S promoter and the E8 promoter</b></center>
+<br />
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