We use Escherichia coli as our chassis,the names of all strains are:Escherichia coli strain Nissle, BL21 Competent E. coli, DH5-Alpha Escherichia coli strain.Our chassis organisms are all widely-used E.coli strains.So they're all very safe, and even if they get out of the lab, the risk can be effectively controlled.

We add lethal sequences to our plasmids, and there are two ways to do that.
SRNA in plasmid 1 is an inhibitory element of lysis2 in plasmid 2, which inhibits lysis2 expression. SRNA in plasmid 2 is an inhibiting element of lysis1 in plasmid 1, which inhibits lysis1 expression. When one of the plasmids is lost, the lytic protein of the other plasmids will not be inhibited, that is, the bacteria will express the lytic protein and cause the bacteria to lyse, thus killing the bacteria that lose the plasmids, so as to prevent the loss of plasmids.
Due to the high concentration of uric acid in the intestines of hyperuricemia patients, we use uric acid as the response signal of the lethal system. When the uric acid concentration exceeds the threshold, HucR is inhibited by uric acid, and Phuco is in the open state. Subsequent genes can be transcribed and translated normally, but since lysis1 is inhibited by sRNA, the bacteria will not be cracked. When the uric acid concentration was lower than the threshold value, HucR inhibited Phuco, and Phuco was turned off. Subsequent genes could not be transcribed and translated, so that sRNA inhibiting lysis2 could not be expressed, while lysis2 could be normally expressed, resulting in death of bacteria. Due to the high concentration of uric acid in the intestinal tract of uric acid patients, the death rate of bacteria in the patient is not high. However, when the bacteria are lost to the external environment, the uric acid concentration in the external environment is lower than the threshold value, which activates the lethal system of the bacteria, leading to the death of bacteria cracking and reaching the purpose and effect of in vitro death.
Death in vitro was controlled by pCold to lysis3. PCold is cold shock plasmid in vitro in 16 ℃ under the condition of using IPTG induction of 3 h can activate lysis3, killed bacteria cracking, achieve the purpose of in vitro to death and effect. PCold came from the IGEM team of Northeastern University, and our two teams had a friendly interlab exchange.

We wear plastic gloves, white coats and goggles when performing the experiment. In addition, the experimental team of our team conducted safety training and had a clear understanding and deep understanding of how to ensure safety during the experiment. During the experiment, we will use the knowledge we received during safety training to ensure safety.
At the same time, we always guarantee the clean and orderly laboratory.

Details

1. According to the Abs results at each time point 0 hours and 6 hours, the cell concentration of Device 1 is significantly lower than that of other devices.
2. According to the fluorescence raw readings, the fluorescence of Device 3 is weakest, the fluorescence of Device 4 is strongest. And the fluorescence of each group increased from 0h to 6 h, abnormally, the fluorescence of the two bacteria of Device 5 was different.
3. There is significant difference in plasmid conversion rate between negative control (device 1) and positive control (device 2).