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| − | <h3 class="sectiontitle">BBa_K2197300
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| − | <h4 class="sectiontitle">Purpose</h4>
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| − | <p style="text-align:left !important;">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.
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| − | <h4 class="sectiontitle">Design</h4>
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| − | <p style="text-align:left !important;">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.
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| − | <h4 class="sectiontitle"> Calibration</h4>
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| − | <p style="text-align:left !important;">graphs
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| − | GFP expression regulated by a concentration-sensitive-promoter (see link)
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| − | http://www.nature.com/nbt/journal/v28/n4/full/nbt.1617.html
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| − | BBa_K2197301
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| − | <p style="text-align:left !important;">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.
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| − | http://www.nature.com/nbt/journal/v28/n4/full/nbt.1617.html
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| | 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. | | 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 </p> | | http://www.nature.com/nbt/journal/v28/n4/full/nbt.1617.html </p> |
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| − | BBa_K2197400
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| − | <div class="divider"></div>
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| − | <h4 class="sectiontitle">Purpose</h4>
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| − | <p style="text-align:left !important;">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.
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| − | </p>
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| − | <h4 class="sectiontitle"> design</h4>
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| − | <p style="text-align:left !important;">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 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.
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| − | http://www.nature.com/nbt/journal/v28/n4/full/nbt.1617.html
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| − | <h3 class="sectiontitle" style="clear:both;">BBa_K2197401</h3>
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| − | <div class="divider"></div>
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| − | <p style="text-align:left !important;">This is a composite part encoding hucO(BBa_K2197303) and smUOX(BBa_K2197402) gene. smUOX is a human gene for urate oxidase, which is an enzyme that digest uric acid. 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 smUOX gene. It is hoped that the expression of the downstream smUOX gene is regulated by the concentration of uric acid in a proportional manner so that uric acid is cleared out.
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| − | http://www.nature.com/nbt/journal/v28/n4/full/nbt.1617.html
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| | </p> | | </p> |
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| − | BBa_K2197500
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| − | <p style="text-align:left !important;">This composite part contain the constitutive promoter J23100, RBS B0034, ygfU K2197501 (an e.coli originated uric acid transporter) and double terminator B0015. The YgfU is constitutively expressed under J23100. It is expected that the YgfU will absorb surrounding uric acid into the cell, while the urate oxidase ( present naturally in E.coli) converts uric acid into Allantoin. This part act as a validation part to check for expression and the function of ygfU. The full assay construct is designated as K2197502.
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| − | BBa_K2197502
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| − | <p style="text-align:left !important;">This part uses the k2197300 construct but the GFP (E0040) is replaced by the YgfU (K2197501) The uric acid-concentration sensitive promoter device K2197300 allows for a negative feedback loop by expressing more ygfU when there is a large amount of uric acid. The higher expression rate of ygfU allows for more uric acid to be absorbed into the e coli, in order to lower the uric acid concentration. This part is use to absorbing excess uric acid produced in the alimentary canal(primarily small intestines).</p>
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| − | BBBa_K2197510
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| − | <div class="divider"></div>
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| − | <p style="text-align:left !important;">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)
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| − | BBa_K2197600
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| − | <p style="text-align:left !important;">This composite part consists of two sessions which is the promoter and BBa_K1456006 (designed by Mustafa Semih Elitok iGEM14_ATOMS-Turkiye). The promoter HucR which is a bacterial transcriptional repressor will dissociates from the binding site hucO8 in the presence of uric acid and allow the expression of BBa_K1456006, Aprotinin. Aprotinin is a potent protease inhibitor which prevents the formation of xanthine oxidase (XO) from xanthine dehydrogenase (XDH). XO is an enzyme that catalyze the oxidation of hypoxanthine to xanthine and can further catalyze the oxidation of xanthine to uric acid. Thus, when the engineered cell sense the presence of uric acid, it will allow the expression of the downstream gene, producing aprotinin, which can stop XDH turning to XO so that neither Xanthine nor uric acid can be formed. </p>
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| − | https://static.igem.org/mediawiki/2017/c/c2/T--Hong_Kong_UCCKE--_Basic_Parts-K2197600.png
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