The goal of our Herb Tasters project is to design biosensors to detect heavy metals and aflatoxin in Chinese medicine. We used synthetic biology technology to make E. coli carry different types of plasmid which can express fluorescence in the presence of metal ions or aflatoxin. The prototype of our product is designed in a form of bio-kit package consisting of these biosensors. Also, we designed our product have biosafety mechanism to prevent of E. coli from spreading and polluting the environment. We conducted experiments to test the functions of biosensors and the effectiveness of biosafety mechanism
Figure. Framework of The Herb Tasters
We designed new parts or used the previously existing parts to detect aflatoxin and three kinds of heavy metals—lead, copper, arsenic ions in Chinese medicine.
1. Aflatoxin: we used aflatoxin metabolism and SOS response to design the circuit. Based on our literature review, we submitted five new basic parts for aflatoxin metabolism and we redo ligation for two composite parts
2. Lead ion: the circuit consists of two parts: generator of the regulator PbrR which regulates the lead resistance operon; lead ion reporter of which the GFP express fluorescence when the dimer of lead ion and PbrR binds onto the PbrR promoter.
For the lead biosensor, we designed an autoregulation mechanism to control the PbrR generation because the GFP expression will be affected when PbrR concentration is higher than lead lion. We submitted three new composite parts.
3. Copper ion: we tried a new application of the device of the copa promoter with a GFP reporter (BBa_K1555000 ; iGEM14_NJAU_China), to detect Cooper ion in Chinese medicine.
4. Arsenic ion: we tried a new application of GFP generator under Ars promoter (BBa_K1106004 ; iGEM13_Buenos_Aires) to detect the arsenic ions in the Chinese medicine.
We conducted two sets of experiments: we tested the survival of the E.coli in Chinese medicine to test the false negative problem (make sure the no expression of GFP is due to E. coli death). We tested the biosensor function through measuring fluorescence in different ion concentration.
Function Assays in Chinese Medicine
We redo all experiments in different Chinese medicine. In addition, considering Chinese herbal medicines are plants and some heavy metal ions are essential micronutrients for their metabolism, we also use EDTA to chelate heavy metal ions containing in our testing Chinese herbs to accurately assess levels of heavy metal among these Chinese medicines.
For the lead biosensor, we designed an autoregulation mechanism to control the PbrR generation because the GFP expression will be affected when PbrR concentration is higher than lead lion. We compared the GFP expression of auto-regulated PbrR generator and the generator under control with a constitutive promoter to model the effect of autoregulation.
Product Biosafety Test: sterilization
We have three steps of biosafety mechanism for our prototype. In one step, we used a special plant (hibiscus) to kill E. coli. Our experiment shows that hibiscus has a great function of bacteria sterilization.