Difference between revisions of "Part:BBa K3179005:Design"

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===Design Notes===
 
===Design Notes===
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This part is constructed by adding 2X Kturn sequence before Adenovirus E1A gene. Due to the existence of 2X Kturn, the translation of E1A is regulated by L7Ae. E1A is an early gene of adenovirus and is essential in the commence of viral replication. This part has an intron which also exists in E1A in the original adenoviral genome. Thus, using the splicing system in eukaryotes can make it function as normal. In our design, 2kt-E1A is used as a response part to startup viral gene expression and lyse target cells.
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This part is constructed by adding a 2X Kturn sequence before the E1A gene and E1B 55K of Ad5. Due to the existence of 2X Kturn, the translation of E1A and E1B 55K is regulated by L7Ae. E1A is an early gene of adenovirus and is essential in the commence of viral replication. E1B 55K can enhance the ability to lyse cells. In our design, we used P2A sequence to link E1A and E1B 55K, so that they can still maintain normal expression levels when using the same promoter. This part has an intron which also exists in E1A of the wild adenovirus. Using the splicing system in eukaryotes can make it function as normal. In our design, 2kt-E1A-E1B 55K is used as a response part to initiate the viral gene expression and lyse target cells.
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We integrate 2kt-E1 with miR885-5p-target and miR663b-target so that while miRNA885-5p and miR663b-target are absent in the cell, E1A can be translated to startup viral gene expression and lyse target cells.
+
 
 +
We integrate 2kt-E1A-E1B 55K with miR885-5p-target and miR663b-target so that while miRNA885-5p and miRNA663b are absent in the cell, E1A can be translated and initiate viral gene expression to lyse target cells.
  
 
===Source===
 
===Source===
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1 Xie Z, Wroblewska L, Prochazka L, et al. Multi-input RNAi-based logic circuit for identification of specific cancer cells[J]. Science, 2011, 333(6047): 1307-1311.
 
1 Xie Z, Wroblewska L, Prochazka L, et al. Multi-input RNAi-based logic circuit for identification of specific cancer cells[J]. Science, 2011, 333(6047): 1307-1311.
 
<br style="clear: both" />2 Saito H, Kobayashi T, Hara T, et al. Synthetic translational regulation by an L7Ae–kink-turn RNP switch[J]. Nature chemical biology, 2010, 6(1): 71.
 
<br style="clear: both" />2 Saito H, Kobayashi T, Hara T, et al. Synthetic translational regulation by an L7Ae–kink-turn RNP switch[J]. Nature chemical biology, 2010, 6(1): 71.
 +
<br style="clear: both" />3 Berk A J. Functions of adenovirus E1A[J]. Cancer surveys, 1986, 5(2): 367-387.
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<br style="clear: both" />4 Martin M E D, Berk A J. Adenovirus E1B 55K represses p53 activation in vitro[J]. Journal of virology, 1998, 72(4): 3146-3154.
 +
<br style="clear: both" />5 Kim J H, Lee S R, Li L H, et al. High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice[J]. PloS one, 2011, 6(4): e18556.

Latest revision as of 16:56, 21 October 2019


2Kt-E1A+E1B55K


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 859
    Illegal PstI site found at 1609
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 1609
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2435
    Illegal BamHI site found at 63
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 859
    Illegal PstI site found at 1609
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 859
    Illegal PstI site found at 1609
    Illegal NgoMIV site found at 333
    Illegal NgoMIV site found at 1328
    Illegal AgeI site found at 69
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 2461


Design Notes

This part is constructed by adding a 2X Kturn sequence before the E1A gene and E1B 55K of Ad5. Due to the existence of 2X Kturn, the translation of E1A and E1B 55K is regulated by L7Ae. E1A is an early gene of adenovirus and is essential in the commence of viral replication. E1B 55K can enhance the ability to lyse cells. In our design, we used P2A sequence to link E1A and E1B 55K, so that they can still maintain normal expression levels when using the same promoter. This part has an intron which also exists in E1A of the wild adenovirus. Using the splicing system in eukaryotes can make it function as normal. In our design, 2kt-E1A-E1B 55K is used as a response part to initiate the viral gene expression and lyse target cells.

We integrate 2kt-E1A-E1B 55K with miR885-5p-target and miR663b-target so that while miRNA885-5p and miRNA663b are absent in the cell, E1A can be translated and initiate viral gene expression to lyse target cells.

Source

Adenovirus protein, get from NCBI and the sequence was synthesized from a biological company.

References

1 Xie Z, Wroblewska L, Prochazka L, et al. Multi-input RNAi-based logic circuit for identification of specific cancer cells[J]. Science, 2011, 333(6047): 1307-1311.
2 Saito H, Kobayashi T, Hara T, et al. Synthetic translational regulation by an L7Ae–kink-turn RNP switch[J]. Nature chemical biology, 2010, 6(1): 71.
3 Berk A J. Functions of adenovirus E1A[J]. Cancer surveys, 1986, 5(2): 367-387.
4 Martin M E D, Berk A J. Adenovirus E1B 55K represses p53 activation in vitro[J]. Journal of virology, 1998, 72(4): 3146-3154.
5 Kim J H, Lee S R, Li L H, et al. High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice[J]. PloS one, 2011, 6(4): e18556.