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.
Team:DLUT China/Safety
Safety
We add lethal sequences to our plasmids, and there are two ways to do that.
Figure 1 Conditional lethal system 1, composed of two plasmids. Plasmid 1 is composed of
the promoter hucO, hypothetical uricase regulator which called HucR, uric acid transporter which called ygfU, sRNA and lysis protein. Plasmid 2 is composed of the sRNA and lysis protein.
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 2 Conditional lethal system 2, composed of one plasmid. Plasmid 3 is composed of
the promoter pCold and lysis protein.
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.
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.
At the same time, we always guarantee the clean and orderly laboratory.
Miniprepped plasmid DNA (over 250ng) have been dried down. They are in 96 well plate format sealed with adhesive foil. We commissioned GenScript Biotech Corp. to carry the transportation components. Submission shipments have been declared and labeled as DNA (non-hazardous, non-regulated, non-infectious, for research purposes only).
We investigated the approval and management of probiotics in different countries.And find that in China, there is no separate regulation or management method for the safety and quality control of edible fungicides, and the quality of products on the market is also uneven.So after the team members’ discussion,we believe that in the future, we can improve relevant regulations in the following aspects：
1)The safety evaluation of probiotics should consider the safety of people with low immunity;
2)How to accurately quantify live probiotics entering and colonizing the intestines;
3)How to simplify laws and regulations to encourage industrial development under the premise of ensuring food safety;
4)How to develop probiotics, product development, and laws and regulations to form a systematic process, and then promote each other and develop together;
5)How to effectively communicate these standards and norms, and build a global system to adapt to the globalization of the probiotic industry.
Insecticide safety of different countries
1)The safety evaluation of probiotics should consider the safety of people with low immunity;
2)How to accurately quantify live probiotics entering and colonizing the intestines;
3)How to simplify laws and regulations to encourage industrial development under the premise of ensuring food safety;
4)How to develop probiotics, product development, and laws and regulations to form a systematic process, and then promote each other and develop together;
5)How to effectively communicate these standards and norms, and build a global system to adapt to the globalization of the probiotic industry.