Part:BBa_K2197300
Expression of GFP under the control of a uric acid concentration-sensitive promoter device
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 light intensity 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.
Mechanism of BBa_K2197300
Expression of a strong repressor (mUTS)
Operator site and GFP
Expression without uric acid (-UA)
mUTS binds with HucO and repress the expression of downstream GFP.
Expression with uric acid (+UA)
mUTS dissociates with HucO to different extents according to the concentration of uric acid, thus expressing downstream GFP at different levels.
Design
Part BBa_K2197300 can be divided into two sessions, the expression of a strong repressor and the GFP. The repressor and a DNA sequence motif upstream the GFP work as a promoter which is sensitive to the concentration of uric acid (UA). 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 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 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.
The design was referenced from an article from Nature, titled "Self-sufficient control of urate homeostasis in mice by a synthetic circuit". We engineered the circuit, which includes hucO, with reference to the article.
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.
GFP expression regulated by a concentration-sensitive-promoter
http://www.nature.com/nbt/journal/v28/n4/full/nbt.1617.html
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 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 - 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 555
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 291
Illegal BsaI.rc site found at 1814
Illegal SapI.rc site found at 255
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