Team:Hong Kong UCCKE/Composite Part

BBa_K2197300

Purpose

Engineered e.coli encodes part BBa_K2197300. By adding blood samples to culture medium where engineered e.coli is cultured, e.coli expresses different levels of GFP. By analysing the GFP level with plate reader, 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[1] 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

Design of BBa_K2197300 Design of BBa_K2197300
Expression of a strong repressor (mUTS) Expression of a strong repressor (mUTS)
Operator site and GFP Operator site and GFP
Expression without uric acid (-UA) Expression without uric acid (-UA)
Expression with uric acid (+UA) Expression with uric acid (+UA)

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

Mechanism of BBa_K2197301Mechanism of BBa_K2197301

BBa_K2197400

Purpose

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[3]. 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_K2197502. 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.

Design

Part BBa_K2197400 can be divided into two sessions, the promoter and the smUOX expression[1]. The promoter is designed to be sensitive to the concentration of uric acid. This promoter control 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, 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 bound 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 smUOX gene and a terminator B1002. 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 control the expression of smUOX. Engineered e.coli encodes part BBa_K2197400. http://www.nature.com/nbt/journal/v28/n4/full/nbt.1617.html

Design of BBa_K2197400 Design of BBa_K2197400
Expression of a strong repressor (mUTS) Expression of a strong repressor (mUTS)
Operator site and smUOX Operator site and smUOX
Expression without uric acid (-UA) Expression without uric acid (-UA)
Expression with uric acid (+UA) Expression with uric acid (+UA)

BBa_K2197500

This composite part contains a constitutive promoter J23100, a RBS B0034, a ygfU(an e.coli originated uric acid transporter) gene[4] and a double terminator B0015. It is expected that the YgfU, a Uric Acid Transporter from Escherichia coli, will absorb surrounding uric acid into the cell, and the urate oxidase in it ( present naturally in E.coli) converts uric acid into Allantoin[3]. This part act as a basic part to check for expression and the validity of ygfU.

Design of BBa_K2197500Design of BBa_K2197500

BBa_K2197502

Purpose

According to our research, one-third of serum uric acid is found in our alimentary canal(primarily small intestines). By enclosing the engineered bacteria (BBa_K2197502) in a capsule, we hope i to absorb excess uric acid produced in the guts. Our ultimate goal is to create a capsule of transformed E.coli with parts BBa_K2197400 and BBa_K2197502. 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[4]. 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.

Design

This composite part can also be divided into two sessions. The uric acid concentration-sensitive promoter[1] and BBa_K2197500. Referring to the mechanism of the promoter that we have explained in the project design page, when there is a large amount of uric acid, the repressor protein mUTS will disassociate from HucO, the operating site, and allow the expression of downstream gene which will express more Ygfu. The higher expression rate of Ygfu allows more uric acid to be absorbed into the E. coli, so that it can lower the uric acid concentration outside the cells.

Design of BBa_K2197502 Design of BBa_K2197502
Expression of a strong repressor (mUTS) Expression of a strong repressor (mUTS)
Operator site and YgFU Operator site and YgFU
Expression without uric acid (-UA) Expression without uric acid (-UA)
Expression with uric acid (+UA) Expression with uric acid (+UA)

BBa_K2197510

This composite part encodes a uric acid and glucose transporter Glut 9b(also known as SLC2A9), a gene originated from homo sapiens. Using the same construct as K2197500, the main gene of interest is swapped to Glut 9b, instead of Ygfu. The Glut 9b is expected to serve the same function as the Ygfu, which is to absorb uric acid into the cell. This is not going to be tested in the assay but rather to use as an demonstration of this idea Incorporated with a human gene(eukaryotic cell) instead of e coli (prokaryotic cells)

Design of BBa_K2197510Design of BBa_K2197510

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

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