Difference between revisions of "Part:BBa K1722010"
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We construct hTERT promoter and tRNA in the same plasmid to produce tRNA. In the unnatural amino acid orthogonal system that we are constructing, hUPll promoter and hTERT promoter can recognise bladder specific RNA polymerase and tumor specific RNA polymerase, respectively. Only when the two promoters are activated simultanuously can both AckRS and tRNA be produced. In this way, Ack can be attached to tRNA that perfectly pair with the mRNA chain to express the protein. | We construct hTERT promoter and tRNA in the same plasmid to produce tRNA. In the unnatural amino acid orthogonal system that we are constructing, hUPll promoter and hTERT promoter can recognise bladder specific RNA polymerase and tumor specific RNA polymerase, respectively. Only when the two promoters are activated simultanuously can both AckRS and tRNA be produced. In this way, Ack can be attached to tRNA that perfectly pair with the mRNA chain to express the protein. | ||
| + | hTERT is 454bp in length. <b>Fig. 1</b> shows the DNA sequence of hTERT is successfully amplified by PCR from psi-Check2 vector. From this electrophoretogram, we can see the brightness of hTERT PCR product is rather high compared with DNA Marker, which indicates that the PCR product of hTERT is in a high concerntration. | ||
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| + | <figure style="text-align: center"><img style="width:30%" src="https://static.igem.org/mediawiki/2015/1/14/Tert_pcr3.png"/><figcaption style="text-align:center"><b>Figure 1.</b> Electrophoretic analysis of PCR produution of hTERT promoter from psi-Check2. <figcaption style="text-align:center">(1:PCR production 2:DL2000 DNA Marker)</figcaption></figure> | ||
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We then performed single digest(EcoRI) and double digest(EcoRI & PstI) to identify our pSB1C3-hTERT-tRNA plasmid.<b>(Fig. 2)</b> From the eletrophoretogram, we have two electrophoresis strips at about 562bp and 2070bp, which are exactly the length of hTERT+tRNA and pSB1C3, respectively in Track 2 and a strip at about 2632bp in Track 3. From this enzyme cutting result, we could make sure the Gene sequence of hTERT+tRNA succeeded in being constructed into pSB1C3 vector. | We then performed single digest(EcoRI) and double digest(EcoRI & PstI) to identify our pSB1C3-hTERT-tRNA plasmid.<b>(Fig. 2)</b> From the eletrophoretogram, we have two electrophoresis strips at about 562bp and 2070bp, which are exactly the length of hTERT+tRNA and pSB1C3, respectively in Track 2 and a strip at about 2632bp in Track 3. From this enzyme cutting result, we could make sure the Gene sequence of hTERT+tRNA succeeded in being constructed into pSB1C3 vector. | ||
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| − | <figure style="text-align: center"><img style="width:30%" src="https://static.igem.org/mediawiki/2015/d/d1/Tert%2Btrna%E9%85%B6%E5%88%87.png"/><figcaption style="text-align:center"><b>Figure | + | <figure style="text-align: center"><img style="width:30%" src="https://static.igem.org/mediawiki/2015/d/d1/Tert%2Btrna%E9%85%B6%E5%88%87.png"/><figcaption style="text-align:center"><b>Figure 2.</b> Identification of recombinant plasmids pSB1C3-hTERT-tRNA by one and two restriction enzymes. <figcaption style="text-align:center">[1:DL2000 DNA Marker 2:pSB1C3-hTERT-tRNA double digest(EcoRI and PstI) 3:pSB1C3-hTERT-tRNA single digest(EcoRI)]</figcaption></figure> |
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Revision as of 06:20, 5 September 2015
hTERT+tRNA Composite
Targeted therapy has become the fastest growing subject in research on cancer treatment. Telomerase, which serves as the tumor marker, is activated in most malignancy while it's negatively expressed in normal cells. Because of the high expression of human telomerase reverse transcriptase(hTERT) gene in telomerase positive cancer cells, researchers construct plasmids with hTERT promoter and effector gene to target at cancer cells and kill them.[1] It has been proved that hTERT can regulate the activation and expression of telomerase in transcription level. Research shows that hTERT promoter is rich in GC but lack of TATA box or CAAT box. It's also methylated and deacetylated in different level.
We construct hTERT promoter and tRNA in the same plasmid to produce tRNA. In the unnatural amino acid orthogonal system that we are constructing, hUPll promoter and hTERT promoter can recognise bladder specific RNA polymerase and tumor specific RNA polymerase, respectively. Only when the two promoters are activated simultanuously can both AckRS and tRNA be produced. In this way, Ack can be attached to tRNA that perfectly pair with the mRNA chain to express the protein.
hTERT is 454bp in length. Fig. 1 shows the DNA sequence of hTERT is successfully amplified by PCR from psi-Check2 vector. From this electrophoretogram, we can see the brightness of hTERT PCR product is rather high compared with DNA Marker, which indicates that the PCR product of hTERT is in a high concerntration.
We then performed single digest(EcoRI) and double digest(EcoRI & PstI) to identify our pSB1C3-hTERT-tRNA plasmid.(Fig. 2) From the eletrophoretogram, we have two electrophoresis strips at about 562bp and 2070bp, which are exactly the length of hTERT+tRNA and pSB1C3, respectively in Track 2 and a strip at about 2632bp in Track 3. From this enzyme cutting result, we could make sure the Gene sequence of hTERT+tRNA succeeded in being constructed into pSB1C3 vector.
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
Both hTERT promoter and tRNA are achieved from Shenzhen Second People's Hospital. We designed primers and amplified the gene sequences from psi-Check2 vector. Using 3A Assembly method, we constructed hTERT and tRNA in pSB1C3.
Source
Both hTERT promoter and tRNA are achieved from Shenzhen Second People's Hospital.
References
[1] Liu Y. Research Progress in Targeting Human Telomerase Reverse Transcriptase (hTERT) Promoter in Cancer Gene Therapy, Journal of Oncology, 15(3): 150-152