Difference between revisions of "Part:BBa K1722012"

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SV40(with Enhancer)+Rluc Composite

SV40+Rluc, the device that we constructed this year, will perform its function with two other devices hUPll+AckRS(BBa_K1722007) and hTERT+tRNA(BBa_K1722010)/ shTERT+tRNA(BBa_K1722011).

SV40 is an abbreviation for Simian Virus 40, a polyomavirus that is found in both monkeys and humans. SV40 promoter, which is one of the earliest virus promoter being found by biologists, can improve the gene expression level of many host cells.^[1] Similar to 35S promoter, SV40 has a relatively small genetic structure and high expression driving ability.^[2-3] As a strong promoter being widely used in genetic engineering,^[4] SV40 has a close affinity with RNA polymerase and can direct the massive synthesizing of mRNA. Different from the SV40 promoter that is already existed in iGEM Distribution kit, this promoter has an enhancer in its sequence.

Rluc can express Renilla luciferase which has become popular as a reporter enzyme for gene expression assays. Renilla luciferase(RLUC) is a blue-light emitting luciferase of marine anthozoan Renilla reniformis.^[5] As a reporter gene, researchers attach it to a regulatory sequence of another gene of interest in bacteria, cell culture, animals or plants. RLUC is chosen as a reporter because the characteristic it confer on organisms expressing it is easily identified and measured. The bioluminescence of the sea pancy, is under the control of a nerve network^[6-8] and is stimulated by changes of intracellular Ca²⁺ concerntration.[9-11] RLUC catalyzes the oxidation of coelenteramide, CO2 and light(480nm）,as in the following scheme:

2015 SZU-iGEM construct SV40(with Enhancer) and Rluc with one codon being amber mutated in the same plasmid. This plasmid, together with two other plasmids, are inserted into the cell. Only when the three plasmids work simultanuously can our orthogonal system behave its function, specifically recognise bladder cancer cells and kill them.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.

Fig. 2 and Fig. 3 shows the electrophoretogram of SV40(with Enhancer) and Rluc being amplified from psi-Check2 by PCR, respectively. The length of SV40 is 419bp and that of Rluc is 936bp. From the two figures we can see the stips of PCR products are exactly in the right site, which means we have successfuly amplified the two gene.

We used primers SV40(up) and Rlu(down) to amplifiy the gene sequence of SV40(with Enhancer) and Rlu from the plasmid we had constructed.(Fig. 2) From the electrophoretogram we can see the electrophoresis strip is in the site of 1355bp, which is exactly the length of SV40(with Enhancer)+Rluc. In this way, we could make sure that we had successfully constructed this device.

We then performed single digest(EcoRI) and double digest(EcoRI & PstI) to identify our pSB1C3-SV40-Rluc plasmid.(Fig. 5) From the eletrophoretogram, we have two electrophoresis strips at about 1355bp and 2070bp, which are exactly the length of SV40+Rlu and pSB1C3, respectively in Track 2 and a strip at about 3425bp in Track 3. From this enzyme cutting result, we could make sure the Gene sequence of SV40+Rlu succeeded in being constructed into pSB1C3 vector.

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. 6)

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. 7.

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

Design Notes

We designed the primers of these two genes and amplified them from the vector psi-Check2, the two genes were then flanked by the iGEM prefix and suffix after amplification. We constructed the two genes in pSB1C3 using 3A Assembly as described in iGEM.org.

Source

We achieved both SV40 promoter and Rluc from Shenzhen Second People's Hospital.

References

[1] Ouy C, Gardnert A, Kao C, et al. A potential of tissue restrictive gene therapy in renal cell carcinoma using MN/CA IX promoter[J]. Anticancer Res, 2005, 25(2A): 881-886

[2] Pu J, Yang Q, Guo D, et al. Effects of nuclear factor of activated T cells on the promoter activity of the constitutively activated SV40[J]. Chinese Journal of Cellular and Molecular Immunology, 2012,28(5): 452-457

[3] Liu QJ, Yin YJ, Wang B, et al. The Activity of SV40 promoter can be inhibited by overexpression of Hemeoxygenase-1 in tumor cells[J]. Cell Biochem Biophys, 2013, 65(3): 287-295

[4] Mcelroy D, Brettell R. Foreign gene expression in transgenic cereals[J]. Trends, Biotechnol, 1994,12(2): 62-68

[5] Jongchan W, Matthew HH, Albrecht G. Structure-function studies on the active site of the coelenterazine-dependent luciferase from Renilla, Proteinscience, 17(10): 725-735

[6] G.H. Parker, Activities of colonial animals. I. Circulation of water in Renilla, J. Exptl. Zool. 31(1920):343–367.

[7] J.A.C. Nicol, Observation on luminescence in Renilla (Pennatulacea), J. Exp. Biol. 32(1955): 299–320.

[8] P.A.V. Anderson, J.F. Case, Electrical activity associated with luminescence and other colonial behaviour in the pennatulid Renilla kollikeri, Biol. Bull. 149(1975): 80–95.

[9] M.J. Cormier, K. Hori, J.M. Anderson, Bioluminescence in coelenterates, Biochim. Biophys. Acta 346(1974):137–164.

[10] J.M. Anderson, M.J. Cormier, Lumisomes: the cellular site of bioluminescence in coelenterates, J. Biol. Chem. 248(1973):2937–2943.

[11] J.M. Anderson, H. Charbonneau, M.J. Cormier, Mechanism of calcium induction of Renilla bioluminescence. Involvement of a calcium–triggered luciferin binding protein, Biochemistry 13(1974): 1195–1200.