Part:BBa_K1722009
| shTERT+GFP Composite | |
|---|---|
| Function | Super Promoter |
| Use in | Cancer cells |
| RFC standard | RFC 10 |
| Backbone | pSB1C3 |
| Submitted by | [http://2015.igem.org/Team:SZU_China SZU_China 2015] |
shTERT+GFP Composite
hTERT, which is short for human telomerase reverse transcriptase, is a cancer-cell specific promoter. It can be activated inside cancer cells with no effect on normal cells. By mutating four base pairs of hTERT sequence, we achieved an improved promoter with higher promote efficiency named super hTERT(shTERT). As an important component of human telomerase, hTERT express only in tumor cells and other immortal cells which are telomerase positive. Only in tumor cells that can express TERT can the promoter being activated to realize targeted expression of effector gene.
Wu S et al. found that the shTERT promoter can enhance the expression of hTERT and maintain its tumor-specific feature.[1] The activity of shTERT promoter detected by luciferase assay was about three times as large as the wild-type hTERT promoter in bladder cancer cells, while it could not be measured in human fiber cells(HFC). According to Zhuang CL et al.(Table 1), the drive efficiency of shTERT promoter was significantly higher than that of wild-type hTERT promoter in bladder cancer cells T24, 5637 and UM-UC-3. Other assays indicated that the synthetic device can significantly inhibit cell growth, decrease motility, and induce apoptosis in bladder cancer cells but not in HFC.[2]
In this plasmid, we construct hTERT with Green Fluorescent Protein(GFP), which is a widely used reporter gene. When shTERT promoter is activated, sfGFP is produced and be oxidized to fluoresce. By inserting this plasmid into T24 and 5637, two lines of bladder cancer cells, we are able to acquire sfGFP. Green fluorescent light is detected using confocal laser scanning microscopy. This indicates that shTERT promoter is able to be activated inside bladder cancer cells.
shTERT is 454bp in length. Fig. 1 shows the DNA sequence of shTERT is successfully amplified by PCR from psi-Check2 vector. From this electrophoretogram, we can see the brightness of shTERT PCR product is rather high compared with DNA Marker, which indicates that the PCR product of shTERT is in a high concerntration.
We then performed single digest(EcoRI) and double digest(EcoRI & PstI) to identify our pSB1C3-shTERT plasmid.(Fig. 2) From the eletrophoretogram, we have two electrophoresis strips at about 1172bp and 2070bp, which are exactly the length of shTERT+sfGFP and pSB1C3, respectively in Track 1 and a strip at about 3242bp in Track 2. From this enzyme cutting result, we could make sure the Gene sequence of shTERT+sfGFP succeeded in being constructed into pSB1C3 vector.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 473
Design Notes
The shTERT promoter that is achieved from Shenzhen Sencond People's Hospital is constructed in psi-Check2 vector. To assemble it with GFP and pSB1C3, we have to design primers with certain restriction enzyme cutting site and amplify it. Then assemble the three parts using 3A Assembly Method.
Source
This composite part is assembled using shTERT and GFP. shTERT promoter is achieved from Shenzhen Second People's Hospital and GFP is achieved from 2015 Distribution Kit. We constructed the two DNA sequence in pSB1C3, the required vector.
References
[1]Zhuang CL, Fu X, Liu L, et al. Synthetic miRNA sponges driven by mutant hTERT promoter selectively inhibit the progression of bladder cancer. Tumor Biol. 2015
[2]Wu S, Huang P, Li C, Huang Y, Li X, Wang Y, et al. Telomerase reverase transcriptase gene promotor mutations help discern the origin of urogenital tumors: a genomic and molecular study. Eur Urol. 2014;65(2):274–7.
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