Difference between revisions of "Part:BBa K2197300"

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Expression of GFP under the control of a uric acid concentration-sensitive promoter device

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 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.
However, the sequence we submitted to the iGEM registry is incorrect Design 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 strong repressor and GFP expression. 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 controls 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 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 to. 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 GFP gene E0040 and a double terminator B0015. As a result mUTS binds to hucO thus 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 controls 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