Composite
hTERT+tRNA

Part:BBa_K1722010

Designed by: Hao Wang   Group: iGEM15_SZU_China   (2015-09-02)

hTERT+tRNA Composite
Function Produce aa-tRNA
Use in Cancer cells
RFC standard RFC 10
Backbone pSB1C3
Submitted by SZU_China 2015

hTERT+tRNA Composite

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).

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. 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.

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.

Figure 1. Electrophoretic analysis of PCR produution of hTERT promoter from psi-Check2.
(1:PCR production 2:DL2000 DNA Marker)

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.

Figure 2. Identification of recombinant plasmids pSB1C3-hTERT-tRNA by one and two restriction enzymes.
[1:DL2000 DNA Marker 2:pSB1C3-hTERT-tRNA double digest(EcoRI and PstI) 3:pSB1C3-hTERT-tRNA single digest(EcoRI)]

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. 3)

Figure 3. Renilla Luminescence

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. 4.

Figure 4. Green fluorescent intensity.
(A: Positive Control Group B: Two Plasmids-no Ack Group C: Two Plasmids-with Ack Group D: Three Plasmids-no Ack Group E: Three Plasmids-with Ack Group

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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE 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


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