Overview

This year, NAU-CHINA has designed a total of 32 parts, including 14 new basic parts and 18 composite parts.

To detect the bile acids levels through dimerization of FXR(BBa_K4164002) and RXRα(BBa_K4164003), we initially built two chimeric proteins composed of an N-terminal CadC and a C-terminal FXR (or RXRα). When the CadC domains dimerize, the downstream CadBA promoter will drive green fluorescent protein (GFP) expression. In the presence of CDCA, the chimeric receptor FXR-CadC and RXRα-CadC undergo ligand-induced dimerization, which allows for the binding of the pCadBA operon region and promotes the transcription of GFP.

Experiments have shown that there is something wrong with above-mentioned pathways. In order to simplify the testing process, our team constructed a more stable gene circuit. We designed BBa_K4164001(BSS) to hydrolyze the 3-sulfate esters of bile acids routinely contained in urine such as CDCA-S. After that, the de-sulfated CDCA-S(CDCA) can activate FXR, which will form a heterodimeric complex with RXRα. The linked ddRFP-A1 and ddRFP-B1 are correspondingly driven to dimerize and exhibit red fluorescence.

This year, we improved the most prevalent part BBa_J04450 which is often used for signal detection. The expression of mRFP1 is expected to be inducible, but we can still observe red fluorescence without using IPTG, which corresponds to strong leak expression. To solve this problem, we improved BBa_J04450 to make the gene produce generate fewer leakage, so that we can construct a more reliable and predictable genetic module for future iGEMers.

If you want to see more details, you can find the usage and characterization in part registry page.

Basic Parts

This year, NAU-CHINA designed 5 new basic parts, aiming to conduct a household liver disease monitoring method in a cell-free system.

BBa_K4164001 BSS is used to express BSS, in order to hydrolyze the the sulfate group of CDCA-S, which are the predominant modification of sulfonated bile acids in urine.

We fused ddRFP-A1 (BBa_K4164004) and ddRFP-B1 (BBa_K4164005) to the C-terminus of FXR (BBa_K4164002) and RXRα (BBa_K4164003) by a 3*GS Linker, respectively. When activated by de-sulfated CDCA, the downstream report module will be triggered, exhibiting a red fluorescent signal visible to the naked eye.

You can click on the part number for more details in the following table.

Composite Parts

To optimize the system and better implement our expected design, we designed 15 composite components by homologous recombination.

We first verified the feasibility of each part separately. Considering the low intensity of BBa_J23105 which exhibited invisible red fluorescent with overnight culture, we also replaced it with T7lac promoter from pET-29a(+) and purified protein to improve our system.

Parts Collection

This year NAU-CHINA team combined 5 genes BSS, FXR, RXRα, ddRFPA1, ddRFPB1 aiming to conduct a rapid home-based detection method for liver disease through synthetic biology methods as a collection.

Originally，to reduce the burden of bacteria and avoid inclusion body formation，we tried low promoter at first. These combinations turn out to be inefficient and invisible according to our experiments. After communicating with other teams and consulting professors, we tried T7lac promoter and chose these parts below as our final gene pathway .