Difference between revisions of "Team:Jilin China/Description"

 
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                 <p>Although trying very hard by many brilliant scientists and doctors for decades, the therapies of cancer were still painful and ineffective for patients in many cases. Among various kinds of tumors, solid tumors, which are also the subjects of our projects, have it own features on destroying people’s health. Solid tumors are visible lumps that can be detected by clinical examination. The vessels inside solid tumors are abnormal structurally and functionally, thus cannot provide adequate oxygen and nutrients. The partial pressure of oxygen in normal tissues is 3.21-8.8kPa, while in the solid tumor the pressure is 1.33-4kPa or less than 0.33kPa. Therefore, the solid tumor cells easily undergo necrosis inside, forming regions with necrosis and lack of oxygen, acute or chronic, weakening the effect of radiotherapy and chemotherapy, while the residual tumor cells become more invasive and metastatic. According to many clinical surveys, solid tumor constitutes the most severe type of cancers that threaten human health.</p>
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                 <p>We were inspired by the pioneer work from the TecMonterrey team of 2013 iGEM competition, which demonstrated that the TAT-Apoptin could kill tumor cells. Furthermore, they used a hypoxia promoter from E. coli to regulate the expression of TAT-Apoptin so that this protein could be expressed in the anaerobic regions, such as regions inside the solid tumors. </p>
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<p>Synthetic biology provides scientists with a novel approach to tackle with cancer cells, which is to use the modified bacteria to express toxic protein as drugs to kill cancer cells yet do no significantly affect the survival of normal cells. The work of Jeff, published in 2016, proved the validity and feasibility of this approach.  In addition to this pioneer work, many kinds of bacteria are regarded safe in vivo. These bacteria show no immunogenic to human bodies, and they could be controlled by the recombinant plasmids. Besides, the growth and reproduction of anaerobic bacteria predominantly happens in hypoxia regions, which coincides with the feature of solid tumors. We took advantage of all these previous work and  planned our own project accordingly.</p>
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                 <p>Although TAT-Apoptin could induce the apoptosis of tumor cells effectively, it seemed that E. coli was not a suitable vehicle to deliver TAT-Apoptin into tumors. We thought that anaerobic microorganisms might be more effective than E. coli to target the tumor tissues. After extensive literature searching and consulting with experts from different research fields, we found that Bifidobacterium longum (B.longum) should be an ideal vehicle to deliver TAT-Apoptin. First, B. longum only grows in anaerobic regions, which means that the expression of TAT-Apoptin from B. longum can only happen in anaerobic regions, such as the anaerobic regions inside solid tumors. More importantly, B. longum is safe to human body since it produces no toxin to the host. Furthermore, it has anti-tumor biological effects. Considering all these information, we have chosen B. longum instead of E.coli to express the TAT-Apoptin.
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                <p>We were inspired by the work of XXX team in 2015 that the modified bacteria with TAT-apoptin expression elements could be used to kill tumor cells. Apoptin is a protein consisting of 121 amino acids and can selectively induce apoptosis of tumor cells yet do no harm to normal cells. It interacts with histone proteins and non-histone proteins in the H1 group in plasmosome and such interaction could lead to DNA rupture and enrichment. TAT was added to enhance the ability of apoptin to enter into the cells.</p>
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                <p>In addition, we learned from other studies that bifidobacterium could act as a safe bacteria in human body, which only replicates in anaerobic region. That is to say, it could preferably reproduce in solid tumors in human bodies. It also have an inhibitory effect on the occurrence and development of a variety of tumors. HU promoter was also constructed into our device to up-regulate the expression of apoptin in bifidobacterium. After that, the sequence of PMB1, which has two orf for bifidobacterium, was constructed into the device to make sure the replication of the plasmid.</p>
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                <p>Once we determined the bacteria and the protein drug to use, we started to construct our device. However, after a long time of experiments, we gradually realized that the protein could not be secreted from the bifidobacterium, since the apoptin formed inclusion bodies in the bacteria. Considering this, we started to investigate the mechanism of secretion in bifidobacterium. We found several signal peptides that could assist secretion. However, the efficiency of these peptides remained unclear in the paper so it was difficult to choose. After long time of discussion, we selected two signal peptides, Tmp1 and Sec2, to add into our device and we decided to test the function of them in our later experiments.The two devices were constructed in the vector, PUC18, for the following steps.
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                <p>To facilitate the expression of TAT-apoptin in B. longum, many biobricks have been replaced or added in the devices. First of all, Tmp1, originally cloned from the plasmid of Bifidobacterium that contains one ori and two orf sequences was added as the first biobrick to regulate the expression of the TAT-apoptin in B. longum. In addition, the promoter and RBS sequence from hup gene of B.longum that could function in the recombinant plasmid inside B. longum was also added to regulate the expression of this protein. To facilitate the secretion of TAT-apoptin, the signal peptides of Sec2 and Tmp1 were added to direct the secretion process of the fused protein in two separate devices. To confirm the function of TAT-Apoptin and extend the work of the TecMonterrey team, the two devices were transformed into B.longum and the animal experiments were done, which showed that our idea is feasible. More details about the experiments are available in our wiki.</p></div>
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Latest revision as of 17:30, 19 October 2016

Particles

Improvement

We were inspired by the pioneer work from the TecMonterrey team of 2013 iGEM competition, which demonstrated that the TAT-Apoptin could kill tumor cells. Furthermore, they used a hypoxia promoter from E. coli to regulate the expression of TAT-Apoptin so that this protein could be expressed in the anaerobic regions, such as regions inside the solid tumors.

Although TAT-Apoptin could induce the apoptosis of tumor cells effectively, it seemed that E. coli was not a suitable vehicle to deliver TAT-Apoptin into tumors. We thought that anaerobic microorganisms might be more effective than E. coli to target the tumor tissues. After extensive literature searching and consulting with experts from different research fields, we found that Bifidobacterium longum (B.longum) should be an ideal vehicle to deliver TAT-Apoptin. First, B. longum only grows in anaerobic regions, which means that the expression of TAT-Apoptin from B. longum can only happen in anaerobic regions, such as the anaerobic regions inside solid tumors. More importantly, B. longum is safe to human body since it produces no toxin to the host. Furthermore, it has anti-tumor biological effects. Considering all these information, we have chosen B. longum instead of E.coli to express the TAT-Apoptin.

Improvement

To facilitate the expression of TAT-apoptin in B. longum, many biobricks have been replaced or added in the devices. First of all, Tmp1, originally cloned from the plasmid of Bifidobacterium that contains one ori and two orf sequences was added as the first biobrick to regulate the expression of the TAT-apoptin in B. longum. In addition, the promoter and RBS sequence from hup gene of B.longum that could function in the recombinant plasmid inside B. longum was also added to regulate the expression of this protein. To facilitate the secretion of TAT-apoptin, the signal peptides of Sec2 and Tmp1 were added to direct the secretion process of the fused protein in two separate devices. To confirm the function of TAT-Apoptin and extend the work of the TecMonterrey team, the two devices were transformed into B.longum and the animal experiments were done, which showed that our idea is feasible. More details about the experiments are available in our wiki.

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