Design

In order to detect and prevent water blooms at early stages, we hope that a single plasmid can be used to express β-galactosidase when C. elegans is under stress state, and make it to turn blue when exposed to certain concentration of microcystin so that the detection could be more efficient. We use existing components with independent functions to connect multiple components into systemic pathways with complete functions to construct complete plasmids to achieve our goal.

We set the stage for building the entire systemic pathway when we built the individual components. Firstly, we use Phsp-6 promoter to replace the original promoter Pbbs-1. It is the mitochondrial stress promoter, and may respond to increased reactive oxygen species (ROS) level caused by microcystin. Then we use modified LacZ to replace original TIR gene located on the downstream of Phsp-6. For the last step, we insert Met+SV40 NLS to guide LacZ gene into the nucleus with the way of homologous recombination.

Build

restriction endonuclease, ligase to construct the pathway, designed primers in strict accordance with the instructions, and carried out the operation process to reconstruct our plasmid. We used PCR technique to obtain gene segments from plasmids containing those sequences, and then verified and purified by gel recovery. Taking the replacement of Phsp-6 promotor as an example. We first used PCR to obtain the linearized sequence segment Phsp-6. The purified carrier and gene segment were verified by re-gel recovery. At last, the carrier and sequence segments were connected by T4 ligase, transformed and cultured. Finally, we validated the colony of bacteria to obtain our plasmid. The replacement of the TIR to modified LacZ was in the same previous way.

We attempted to ligate SV40 NLS directly to the carrier into complete plasmids using homologous recombinases at the last step. Firstly, we used PCR to obtain the linearized carrier Phsp-6: LacZ: SL2: mcherry and the segments. Then gel recovery was used to verify the purification of the carrier and fragments. Finally, homologous recombinase was used to directly link the linearized carrier.

Result

We try to use the reporter gene GFP, it can show whether the promoter hsp-6 works under stimulation of the MC-LR. In that case, we put the C. elegans under a fluorescence microscope to test the strength of the fluorescence and used software to calculate the intensity. At first, we designed 3 periods (each 24 hours) to check the fluorescence (Fig.1).

Learn

Experimental operation has a great influence on the experiment. In the process of picking C. elegans, they cannot be put under stress. C. elegans under stress will emit fluorescence even if C. elegans do not be stimulated by microcystin, thus interfering with the experiment. However, we can use the promoter hsp-6 which can respond to increased ROS levels caused by MC-LR (microcystin), and it can play a role in other circumstances. As we have mentioned before, the current experiment does have some wobbles, but we believe that even after the iGEM competition, we will continue to promote our project, make efforts to use synthetic biology to improve the world environment and make rational use of resources.