|Use in||Cancer cells|
|RFC standard||RFC 10|
|Submitted by||SZU_China 2015|
hTERT+tRNA, the device that we constructed this year, will perform its function with two other devices hUPll+AckRS(BBa_K1722007) and SV40+Rlu(BBa_K1722012).
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. 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.
We constructed another gene downstream of shTERT called tRNA, which encodes an unusual transfer RNA(tRNA) with a CUA anticodon. The tRNA(CUA) that is produced can be charged with Acetyllysine(Ack) with the help of Acetyllysine tRNA synthetase(AckRS). It is also the first step in translating UAG amber codon.
In our project, when shTERT is activated inside bladder cancer cell, downstream tRNA sequence can be expressed to produce tRNA with CUA as its anticodon. Together with AckRS, tRNA can carry Ack and pair with the amber UAG stop codon of the mRNA chain to completely produce the interest protein.Our unnatural amino acid orthogonal system consists of three devices(plasmids).
(1)hUPll+AckRS(BBa_K1722007): hUPll is a bladder-cell specific promoter. when it's activated, AckRS, a tRNA synthetase, will be produced.
(2)hTERT+tRNA(BBa_K1722010)/ shTERT+tRNA(BBa_K1722011): hTERT and shTERT are cancer-cell specific promoters. tRNA can be expressed out when the promoter is activated.
(3)SV40+Rluc(BBa_K1722012): SV40 is a widely used strong promoter. Rluc is a reporter that can produce RLUC which is a kind of luciferase.
There is an amber stop codon UAG in the sequence of Rluc. The tRNA that is produced from the second plasmid has CUA as its anticodon, which can pair with the stop codon of the mRNA chain of Rluc. AckRS can achieve the attachment of Ack, the unnatural amino acid, and the tRNA. In this way, when all the three promoters are activated inside bladder cancer cell, Ack can be charged to the specific tRNA and then the anticodon of tRNA can pair with the stop codon on the mRNA chain of Rluc. In natural condition, this Rluc gene cannot be fully expressed because of the amber stop codon. However, with our orthogonal system, it can be produced and detected.
The three plasmids that we've constructed were inserted into three lines of cells(HFC, Hela and 5637). HFC is short for Human Fiber Cells, which is a kind of normal cell in human bladder. Hela and 5637 are cervical cancer cell line and bladder cancer cell line, respectively. We set two groups in each cell lines, one have Ack, an unnatural amino acid, in the culture medium, and another do not have it. The luminescence intensity of 5637 with Ack in the medium is much higher than other groups, which indicates that the specificity of the two promoters are high enough and our orthogonal system is working.(Fig. 2)
We also constructed two plasmids and three plasmids system with GFP as their reporter gene. These systems were transfected into T24, a kind of bladder cancer cell. The working efficiency of our orthogonal system can be detected by comparing the luminescence intensity of green fluorescent of each groups. Two plasmids system is composed of hUPll-AckRS-GFP(amber mutated) and hTERT-tRNA. And the construction members of three plasmids system include hUPll-AckRS, hTERT-tRNA and SV40-GFP(amber mutated). Each system is divided into two groups: one have Ack in the medium and another one do not have Ack. Adding a positive control group, which is constructed by hUPll-GFP(wild-type), we have five groups of bladder cancer cells in total. Luminescent intensity results are shown in Fig. 3.
As we can see, no cell in Two Plasmids-no Ack Group and Three Plasmids-no Ack Group produce green fluorescent light. In Two Plasmids-with Ack Group and Three Plasmids-with Ack Group, however, is full of luminescent cells. From this result, we can tell our orthogonal system work efficiently.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21COMPATIBLE WITH RFC
- 23COMPATIBLE WITH RFC
- 25COMPATIBLE WITH RFC
- 1000COMPATIBLE WITH RFC
Both shTERT 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.
Both shTERT promoter and tRNA are achieved from Shenzhen Second People's Hospital.
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
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.