Part:BBa_K2505005:Design
(tra box)7-CMVmin-atipt4-IVS-IRES-log1-polyA
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1442
- 21INCOMPATIBLE 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 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1220
- 1000INCOMPATIBLE 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
Kakimoto, T. (2001) Identification of plant cytokinin biosynthetic enzymes as dimethylallyl diphosphate:ATP/ADP isopentenyltransferases. Plant Cell Physiol. 42:677-85.
Kurakawa, T., Ueda, N., Maekawa, M., Kobayashi, K., Kojima, M., Nagato, Y., ... Kyozuka, J. (2007). Direct control of shoot meristem activity by a cytokinin-activating enzyme. Nature, 445(7128), 652-655.
Suzuki, T., Miwa, K., Ishikawa, K., Yamada, H., Aiba, H. and Mizuno, T. (2001) The Arabidopsis Sensor His-kinase, AHK4, Can Respond to Cytokinins. Plant Cell Physiol. 42: 107-113.
Spíchal, L., Rakova, N.Y., Riefler, M., Mizuno, T., Romanov, G.A.,Strnad, M. and Schmülling, T. (2004) Two Cytokinin Receptors of Arabidopsis thaliana, CRE1/AHK4 and AHK3, Differ in their Ligand Specifity in a Bacterial Assay. Plant Cell Physiol. 45: 1299-1305.
Petra Neddermann, Cesare Gargioli, Ester Muraglia, Sania Sambuncini, Fabio Bonelli, Raffaele De Francesco, Riccardo cortese (2003) A novel, inducible, eukaryotic gene expression system based on the quorum-sensing transcription facter TraR. EMBO reports VOL 4: 159-165.