Difference between revisions of "Team:Hong Kong UCCKE/Basic Part"

BBa_K2197300

Purpose

Engineered e.coli encodes part BBa_K2197300. By adding blood samples to culture medium which engineered e.coli is cultured, e.coli expresses different level of GFP. By analysing the GFP level with HPLC, the uric acid concentration of the sample can be estimated. This part ensures a rapid detection of uric acid concentration thus gout.

Design

Part BBa_K2197300 can be divided into two sessions, the promoter and the GFP expression. The promoter is designed to be sensitive to the concentration of uric acid. This promoter control the expression of GFP that is downstream the promoter. The promoter session consists of a constitutive promoter J23100, a RBS B0034, a strong repressor KRAB-HucR, a double terminator B0015. HucR is 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 binded to hucO, the expression of downstream gene is restricted according concentration of substrates. 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 GFP gene E0040 and a terminator B1002. As a result mUTS binds to hucO and GFP is not expressed when uric acid is absent or at very low concentration. Alternatively, the complex detaches from hucO and GFP is expressed according to the concentration of uric acid. The promoter control the expression of GFP. Engineered e.coli encodes part BBa_K2197300.

Calibration

graphs GFP expression regulated by a concentration-sensitive-promoter (see link) http://www.nature.com/nbt/journal/v28/n4/full/nbt.1617.html

BBa_K2197301

This is a composite part encoding hucO(BBa_K2197303) and GFP(E0040) gene. 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 allowing expression of a downstream GFP gene. It is hoped that the expression of the downstream GFP gene is regulated by the concentration of uric acid in a proportional manner. -- http://www.nature.com/nbt/journal/v28/n4/full/nbt.1617.html

BBa_K2197302

This is a subpart of BBa_K2197300. Engineered E.coli encodes part BBa_K2197302, which expresses a repressor protein with KRAB amplification. 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 the research, the expression of the downstream gene is regulated by the concentration of uric acid. http://www.nature.com/nbt/journal/v28/n4/full/nbt.1617.html

BBa_K2197303

This is a subpart of BBa_K2197300. Engineered E.coli encodes part BBa_K2197303 which is a operator site for chimeric mammalian urate-dependent transsilencer (mUTS) or KRAB-HucR protein complex. 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 allowing expression of a downstream gene. According the research, the expression of the downstream gene is regulated by the concentration of uric acid. In the reference research article form ResearchGate, hucO motif originated 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. http://www.nature.com/nbt/journal/v28/n4/full/nbt.1617.html

BBa_K2197400

Engineered E.coli encodes part BBa_K2197400. By adding blood samples to culture medium which engineered E.coli is cultured, E.coli expresses different level 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 secrete different amount of smUOX accordingly. It is hoped that most uric acid is digested by the smUOX secreted.