Difference between revisions of "Part:BBa K1722006"
 
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__NOTOC__
 
__NOTOC__
 
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{| style="color:black" cellpadding="5" cellspacing="1" border="3" align="right"
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! colspan="2" style="background:#d7474e;"|SV40(with enhancer)
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|-
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|'''Function'''
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|Promoter
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|-
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|'''Use in'''
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|Mammalian cells
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|-
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|'''RFC standard'''
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|[https://parts.igem.org/Help:Assembly_standard_10 RFC 10]
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|'''Backbone'''
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|[https://parts.igem.org/Help:Plasmid_Backbones pSB1C3]
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|'''Submitted by'''
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|[http://2015.igem.org/Team:SZU_China SZU_China 2015]
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=====<partinfo>BBa_K1722006 short</partinfo>=====
  
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Different from the SV40 promoter distributed by iGEM13_Freiburg(BBa_K1150011), the promoter we are submitting this year includes an enhancer sequence(74bp) upstream of the promoter gene.<b>(Fig. 1)</b>
  
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<figure style="text-align: center"><img style="width:70%" src="https://static.igem.org/mediawiki/2015/c/ce/SV40_sequence.png"/><figcaption style="text-align:center"><b>Figure 1.</b> Sequence of SV40 promoter(with enhancer)<figcaption style="text-align:center"> The 74bp marked in blue is the sequence of the enhancer. </figcaption></figure>
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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.<sup>[1]</sup> Similar to 35S promoter, SV40 has a relatively small genetic structure and high expression driving ability.<sup>[2-3]</sup> As a strong promoter being widely used in genetic engineering,<sup>[4]</sup> SV40 has a close affinity with RNA polymerase and can direct the massive synthesizing of mRNA.
 
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.<sup>[1]</sup> Similar to 35S promoter, SV40 has a relatively small genetic structure and high expression driving ability.<sup>[2-3]</sup> As a strong promoter being widely used in genetic engineering,<sup>[4]</sup> SV40 has a close affinity with RNA polymerase and can direct the massive synthesizing of mRNA.
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We then performed double digest(EcoRI & PstI) and single digest to identify our pSB1C3-hUPll plasmid. From the eletrophoretogram, we have two electrophoresis strip at about 419bp and 2070bp, which are exactly the length of SV40 and pSB1C3 in Track 1 and two strips at about 2489bp in Track 2 and 3. From this enzyme cutting result, we could make sure the Gene sequence of SV40 succeeded in being constructed into pSB1C3 vector.
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<figure style="text-align: center"><img style="width:40%" src="https://static.igem.org/mediawiki/2015/5/50/SV40%28Enhancer%29_%E5%8D%95%E5%8F%8C%E9%85%B6%E5%88%87.png"/><figcaption style="text-align:center"><b>Figure 3.</b> Identification of recombinant plasmids pSB1C3-SV40 by one and two restriction enzymes. <figcaption style="text-align:center">[1:pSB1C3-SV40 double digest(EcoRI & PstI) 2:pSB1C3-SV40 single digest(EcoRI) 3:pSB1C3-SV40 single digest(PstI) 4:DL2000 DNA Marker]</figcaption></figure>
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===

Latest revision as of 14:20, 17 September 2015

SV40(with enhancer)
Function Promoter
Use in Mammalian cells
RFC standard RFC 10
Backbone pSB1C3
Submitted by [http://2015.igem.org/Team:SZU_China SZU_China 2015]
SV40（with Enhancer） is a widely used strong promoter.

Different from the SV40 promoter distributed by iGEM13_Freiburg(BBa_K1150011), the promoter we are submitting this year includes an enhancer sequence(74bp) upstream of the promoter gene.(Fig. 1)

Figure 1. Sequence of SV40 promoter(with enhancer)
The 74bp marked in blue is the sequence of the enhancer.

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.

In our project, we construct SV40 and Renilla luciferase(Rlu) in the same plasmid. By detecting the expression of Rlu, we are able to tell the working efficiency of our system.

SV40 is 419bp in length. Fig. 2 shows the DNA sequence of SV40 is successfully amplified by PCR from psi-Check2 vector. In this electrophoretogram, Track 1,2 and 3 are all SV40 that are amplified. We can see the brightness of SV40 PCR product is rather high compared with DNA Marker, which indicates that the PCR product of SV40 is in a high concerntration.

Figure 2. Electrophoretic analysis of PCR produution of SV40 promoter from psi-Check2.
(1,2,3:PCR production 4:DL2000 DNA Marker)

We then performed double digest(EcoRI & PstI) and single digest to identify our pSB1C3-hUPll plasmid. From the eletrophoretogram, we have two electrophoresis strip at about 419bp and 2070bp, which are exactly the length of SV40 and pSB1C3 in Track 1 and two strips at about 2489bp in Track 2 and 3. From this enzyme cutting result, we could make sure the Gene sequence of SV40 succeeded in being constructed into pSB1C3 vector.

Figure 3. Identification of recombinant plasmids pSB1C3-SV40 by one and two restriction enzymes.
[1:pSB1C3-SV40 double digest(EcoRI & PstI) 2:pSB1C3-SV40 single digest(EcoRI) 3:pSB1C3-SV40 single digest(PstI) 4:DL2000 DNA Marker]
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

We designed the following primers and amplified SV40 promoter from the vector psi-Check2:

By incorporating these primers into hUPII promoter, the promoter is flanked by the iGEM prefix and suffix after amplification.

Source

SV40 promoter with enhancer was achieved 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