This year, NWU-CHINA-A transformed two recombinant plasmids pET28a(+)-ChuA-HrtR and pSB1C3-HtrO-cjBlue into non- pathogenic and edible probiotic E.coli Nissle 1917 which was used to make a biosensor for detecting intestinal micro bleeding. When the probiotic is given to a person with micro intestinal bleeding, it will transport the heme in the intestine to the cells, activating the expression of reporter genes in the bacteria, so as to realize the detection of intestinal micro bleeding.
Our genetic circuit is composed of two parts, one part for transporting heme, and the other part can activate the expression of reporter genes by the heme in bacteria. The expression of pigment proteins color is used to judge the micro bleeding in the intestine. When heme is transported into the cell by ChuA protein, it will interact
with heme binding protein (HrtR protein) in the cell to change the conformation of the heme binding protein, thus relieve its inhibitory effect on the promoter HrtO and express the reporter gene cjBlue to visualize the results.
We selected pET28a (+) with high efficiency as the plasmid vector and
obtained the sequences of ChuA gene and HrtR gene from NCBI and literature,
the expression vector pET28a (+) - ChuA-HrtR was successfully constructed by
using BamHI, EcoRI, NotI and XhoI as the digestion sites.
We found the gene sequence of the promoter HrtO from NCBI and digested it
into the plasmid pSB1C3-cjBlue from iGEM kit, and successfully constructed
the expression vector pSB1C3-HtrO-cjBlue.
We have designed a hardware device for collecting and culturing recombinant
probiotics with low environmental requirements and household use. It mainly
consists of a filter device and a culture device. The filtration device is
used to collect probiotics from feces. The culture device which contains dry
powder medium is used to culture probiotics from the intestine. The
probiotics entering the culture device can express pigment proteins after
being cultured by oscillating and heating.
click here to hardware
Considering the leakage of bacterial strain, we designed a suicide switch.
We envision a blue light activated suicide switch, where we insert nMag and
pMag into different domains of Cas9, and they will form a dimer under the
activation of blue light to form a complete Cas9, and then cut the target
gene fragment under the guidance of gRNA.
