In order to prevent the loss of our Nissle escherichia coli to the external environment, compete with the dominant local strains, and disrupt the normal ecological balance of the environment, we decided to set up in vitro lethal system. Since we were involved in more sequences, we decided to design two plasmids and introduce them into one bacterium at the same time. Therefore, we also set up the plasmids loss killing system.
We designed two systems.
Figure 3 The Figure above is plasmid dlut1, mainly used to express uric acid oxidase; Here is plasmid dlut2, which is responsible for lethal systems.
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.
Figure 4 Above is plasmid dlut3, mainly used to express uric acid oxidase; Here is plasmid dlut4, responsible for lethal systems.
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. In other words, the plasmid loss lethal mechanism of the system is the same as that of system 1.
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.