Part:BBa_K2197400
Expression of smUOX under the control of a uric acid concentration-sensitive promoter device
Purpose Engineered E.coli encodes part BBa_K2197400. By adding blood samples to culture medium where engineered E.coli is cultured, E.coli expresses different levels of human urate oxidase (smUOX), which digests uric acid. The part BBa_K2197400 works with the part BBa_K2197500. The ultimate goal is to create a capsule of transformed E.coli with parts BBa_K2197400 and BBa_K2197500. When the capsule is taken into the human body, E.coli in the capsule absorbs uric acid from the digestive canal with aid with the transporter protein expressed by part BBa_K2197500 (see part BBa_K2197500 description for details). After absorbing the uric acid in the digestive canal, part BBa_K2197400 senses the level of uric acid and then secretes different amount of smUOX accordingly. It is hoped that most uric acid is digested by the smUOX secreted.
Design of BBa_K2197400
Expression of a strong repressor (mUTS)
Operator site and smUOX
Expression without uric acid (-UA)
mUTS binds with HucO and repress the expression of downstream smUOX.
expression with uric acid (+UA)
mUTS dissociates with HucO to different extents according to the concentration of uric acid, thus expressing downstream smUOX at different levels.
Design
Part BBa_K2197400 can be divided into two sessions, the promoter and the smUOX expression. The promoter is designed to be sensitive to the concentration of uric acid. This promoter controls the expression of smUOX that is downstream the promoter. The promoter session consists of a constitutive promoter J23100, a RBS B0034, a strong repressor KRAB-HucR and a double terminator B0015. HucR is in itself a repressor. Its repressing ability is enhanced by KRAB. The resulting repressor is a chimeric mammalian urate-dependent transsilencer (mUTS). hucO is an operative site for mUTS to bind. When mUTS is bound to hucO, the expression of downstream gene is restricted according to the concentration of the substrate. The presence of uric acid limits the binding of mUTS to hucO. The limitation varies as the concentration of uric acid. Downstream of the promoter session is a constitutive promoter J23106, a RBS B0034, a smUOX gene and a double terminator B0015. As a result, mUTS binds to hucO and smUOX is not expressed when uric acid is absent or at very low concentration. Alternatively, the complex detaches from hucO and smUOX is expressed according to the concentration of uric acid. The promoter controls the expression of smUOX. Engineered e.coli encodes part BBa_K2197400.
When this part is put in a composite part, it helps digest uric acid by secreting different amounts of enzymes when uric acid of different concentrations are detected.
Kemmer, Christian & Gitzinger, Marc & Daoud-El Baba, Marie & Djonov, Valentin & Stelling, Jörg & Fussenegger, Martin. (2010). Self-sufficient control of urate homeostasis in mice by a synthetic circuit. Nature biotechnology. 28. 355-60. 10.1038/nbt.1617.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 1813
Illegal PstI site found at 2035 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal NheI site found at 1098
Illegal NheI site found at 1121
Illegal NheI site found at 1906
Illegal PstI site found at 1813
Illegal PstI site found at 2035 - 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 555
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 1813
Illegal PstI site found at 2035 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 1813
Illegal PstI site found at 2035 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 291
Illegal BsaI.rc site found at 1174
Illegal SapI.rc site found at 255
Illegal SapI.rc site found at 2124
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