Team:Worldshaper-Shanghai/Design

Design

Our goal is to inspect the blood uric acid efficientively. Fortunately, we have obtained a uric acid-related HucR protein and pHucO promoter in the literature[1]. Since we thought that an additional fluorescent protein should be added to HucR so that the signal can be collected by a spectrometer, we decided to fuse a mCherry with the HucR.

Produce HucR-mCherry Fusion Protein

First thing first, we should test the relationship among uric acid, HucR-mCherry, and pHucO. The most common way to do this is the Electrophoretic Mobility Shift Assay (EMSA), we designed a gene pathway to produce the HucR-mCherry and synthesized a biotin-labeled pHucO following the EMSA protocol. The gene pathway “HucR-mCherry-His” (Figure 1) contains a strong constitutive promoter (BBa_J23100), RBS (BBa_J34801), HucR (BBa_K3311000) and mCherry (BBa_J06504), following two transcription terminators (BBa_B0010+BBa_B0012), all parts were linked and constructed to pSB1C3. Further research is showed in the results section.

Figure 1 Schematic diagram of HucR-mCherry-His
Figure 1 Schematic diagram of HucR-mCherry-His

Test Paper Kit Design

The Test Paper Kit design is that we mainly focused on. We plan to use positively charged cellulose acetate membrane as the material of the test paper, which binds with pHucO spontaneously due to the Coulomb force under UV, then the HucR-mCherry complexes are bound to the pHucO molecules on the surface of the test paper (Figure 2). Theoretically, after the test paper is dipped in the liquid that contains uric acid, the structure of HucR changes, causing the HucR-mCherry complexes to leave the test paper. Thus, the red fluorescence of the test paper will decrease under excitation source, then a camera is installed for recording images of the test paper that will be analyzed on a computer. More details were showed in Applied design .

Figure 2 Schematic diagram of the test paper design.
Figure 2 Schematic diagram of the test paper design.

Improved Design--Magnetic Beads Kit

Another design depend on the streptavidin magnetic beads. Any biotin-labeled molecule can be bound through the high affinity between streptavidin and biotin. We planed to combine biotin-labeled pHucO oligos, streptavidin magnetic beads and HucR-mCherry (Figure 3). When uric acid is present, HucR-mCherry will leave pHucO. Thus the solution will turn red due to the red fluorescent protein. More details were showed in Applied design .

Figure 3 Schematic diagram of streptavidin- biotin magnetic beads kit.
Figure 3 Schematic diagram of streptavidin- biotin magnetic beads kit.