Part:BBa_K1722012

SV40(with Enhancer)+Rluc Composite

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.

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.

Figure 2. Electrophoretic analysis of PCR produution of SV40 promoter from psi-Check2.

(1,2,3:PCR production 4:DL2000 DNA Marker)

Figure 3. Electrophoretic analysis of PCR produution of Rluc from psi-Check2.

(1:DL2000 DNA Marker 2:PCR production)

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.

Figure 4. Electrophoretic analysis of PCR produution of SV40(with Enhancer)+Rlu from pSB1C3.

(1:DL2000 DNA Marker 2,3,4:PCR production)

We then performed single digest(EcoRI) and double digest(EcoRI & PstI) to identify our pSB1C3-SV40-Rluc plasmid.(Fig. 3) 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.

Figure 3. Identification of recombinant plasmids pSB1C3-Rluc by one and two restriction enzymes.

[1:pSB1C3-Rluc double digest(EcoRI and PstI) 2:pSB1C3-Rluc single digest(EcoRI) 3:DL2000 DNA Marker]

Sequence and Features BBa_K1722012 SequenceAndFeatures ===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.