Composite

Part:BBa_K2505005:Design

Designed by: Hazuki Hasegawa   Group: iGEM17_TokyoTech   (2017-10-16)
Revision as of 20:28, 31 October 2017 by TakumaY (Talk | contribs)


(tra box)7-CMVmin-atipt4-IVS-IRES-log1-polyA


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1442
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 26
    Illegal BglII site found at 72
    Illegal BglII site found at 118
    Illegal BglII site found at 164
    Illegal BglII site found at 210
    Illegal BglII site found at 256
    Illegal BglII site found at 302
    Illegal BamHI site found at 476
    Illegal XhoI site found at 2728
    Illegal XhoI site found at 2740
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1220
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1362


Design Notes

These two genes(apipt4 and log1) are derived from Arabidopsis thaliana and encode enzymes necessary for synthesizing iP (isopentenyladenine) in mammalian cells. iP is a kind of cytokinins that are signaling molecules (Phytohormones) in plants and play important roles in cell growth and differentiation. When these genes are introduced to human cells, EA.hy926, the cells produce iP heterologously. AtIPT4 has the adenylate dimethylallyltransferase ([http://www.genome.jp/dbget-bin/www_bget?ec:2.5.1.112 [EC:2.5.1.112] ]: cytokinin synthase) activity and catalyzes the transfer of an isopentenyl group from dimethylallyl diphosphate (DMAPP) to ATP and ADP, producing cytokinin nucleotides [1]. Note that cytokinin nucleotides are the immature form. LOG1 has the phosphoribohydrolase activity and converts inactive cytokinin nucleotides to the biologically active free-base forms [2].

The expression of these genes are induced by a chemeric transcription factor which consist of eukaryotic activation domain of NF-κB p65 and prokaryotic transcription factor, TraR. The DNA binding domain of TraR binds to tra box in the presence of 3OC8AHL molecules and then CMV minimal promoter will be activated by the activation domain of NF-κB p65 [3].

The DNA sequences of these genes are optimized for expressing in human cells considering the codon usage.


Source

These iP synthesis genes were derived from arabidopsis and tra box was derived from agrobacterium.

References

[1] Identification of plant cytokinin biosynthetic enzymes as dimethylallyl diphosphate:ATP/ADP isopentenyltransferases, 2001. Kakimoto T.

[2] Direct control of shoot meristem activity by a cytokinin-activating enzyme, 2007. Kurakawa T. et. al

[3] A novel,inducible,eukaryotic gene expression system based on the quorum-sensing transcription factor TraR, 2003. Neddermann P. et. al