Team:Hong Kong UCCKE/Basic Part
BBa_K2197302
This is a subpart of BBa_K2197300. Engineered E.coli encodes part BBa_K2197302, which expresses a repressor protein with KRAB amplification[1]. Bacterial transcriptional repressor (HucR) was engineered to be a stronger repressor by fusing it to the C terminus of the Krueppel-associated box (KRAB) protein domain15. The resulting repressor is a chimeric mammalian urate-dependent transsilencer (mUTS). HucR binds a DNA sequence motif (hucO) in the absence of uric acid. When uric aicd is present, HucR dissociates from DNA, thereby allowing expression of a downstream gene. According to our research, the expression of the downstream gene is regulated by the concentration of uric acid.
Pathway from purine to Uric acid
BBa_K2197303
This is a subpart of BBa_K2197300. Engineered E.coli encodes part BBa_K2197303 which is an operating site for chimeric mammalian urate-dependent transsilencer (mUTS) or KRAB-HucR protein complex[1]. The circuit uses a bacterial transcriptional repressor (HucR) that binds a DNA sequence motif (hucO) in the absence of uric acid. When uric acid is present, HucR dissociates from hucO motif, thereby allows an expression of a downstream gene. According to our research, the expression of the downstream gene is regulated by the concentration of uric acid. In a reference research article form ResearchGate, HucO motif originating from Deinococcus radiodurans R1 is replicated 8 times so that the chance of binding is higher. However, this design is very difficult to be synthesized chemically. Therefore, we reduce it to one tandem hucO.
HucO
BBa_K2197402
This is a subpart of BBa_K2197400. The part expresses smUOX[1][2], a mammalian version of urate oxidase. In the human purine and uric acid pathway, purine is converted to uric acid through a series of steps in the purine pathway. Uric acid forms crystals when high concentration is accumulated. Urate oxidase breaks down uric acid into allantoin so that the crystals can be excreted[3].
smUOX
BBa_K2197501
This basic part encodes an E.coli-originated uric acid transporter, Ygfu[4], that transports surrounding uric acid(urate) into the cell and allows the urate oxidase to catalyze uric acid into allantoin[3]. According to other research, ygfu is a proton-gradient dependent, low-affinity (Km 0.5 mm), and high-capacity transporter for uric acid. The sequence was obtained from NCBI [5] and was optimize for IDT Gene Block DNA synthesis.
Ygfu
BBa_K2197511
This basic part encodes a uric acid and glucose transporter Glut 9b(also known as SLC2A9), a gene originated from homo sapiens.
Glut9b
Reference
[1]: Christian Kemmer, Marc Gitzinger, Marie Daoud-El Baba, Valentin Djonov, Jorg Stelling & Martin Fussenegger (2010) Self-sufficient control of urate homeostasis in mice by a synthetic circuit. Nature Biotechnology doi: 10.1038/nbt.1617
[2]: Masako Oda, Yoko Satta, Osamu Takenaka, and Naoyuki Takahata (2002) Loss of Urate Oxidase Activity in Hominoids and its Evolutionary Implications. Molecular Biology and Evolution, Volume 19, Issue 5, 1 May 2002, Pages 640–653
[3]: Ricardo Percudani, Claudia Folli, Ileana Ramazzina (2007) Method for conversion of uric acid to allantoin and related enzymes. WO2007052326 A2
[4]: Papakostas K1, Frillingos S. (2012) Substrate Selectivity of YgfU, a Uric Acid Transporter from Escherichia coli. J Biol Chem. 2012 May 4; 287(19): 15684–15695. doi: 10.1074/jbc.M112.355818
[5]: Gene from NCBI with link: https://www.ncbi.nlm.nih.gov/gene/7159046
