Team:Mingdao/Safety
Mingdao
OUR GOAL
For this project, we’ve genetically modified E. coli Nissle and S. mutans with pyruvate oxidase (SpxB) of S. sanguinis, which can produce H2O2 to antagonize WT S. mutans found in dental caries. To protect GM strains producing H2O2, we utilized an existing part of catalase (KatG) to decompose H2O2 into water and oxygen. In addition, we added the aquaporin gene (AQP) of S. cristatus into GM strains to facilitate H2O2 permeation across the cell membrane. Finally, we developed a homemade candy with probiotics and applied it as our prototype to the real world.
APPLICATION IN REAL WORLD
Chassis
E. coli Nissle (EcN) is a nonpathogenic bacterium isolated by Alfred Nissle in 1917. Since then, many researches used and engineered the strain as a probiotic or therapeutic agent. Dr. Divya Pandya also mentioned in a review paper entitled “Benefits of Probiotics in Oral cavity “ and pointed out E. coli Nissle species is potential used as probiotics in oral cavity with a non-lactic acid producing feature.
Parts
The device we created contains pyruvate oxidase (SpxB), aquaporin (AQP) and catalase (KatG) to produce H2O2, facilitate the transport and detoxify the oxidative stress. All of the three parts are normally and widely present in bacterial and mammalian cells and also in the oral environment. Therefore, we believed they are very safe and have no concerns about any risk to human or the environment.
Safety Test
Image the application in the real world, the candy we made contains E. coli Nissle cells, which will be released when dissolved in the mouse. Prior to reaching to the intestine and be absorbed by tissues, they have to pass through the stomach and incubate at the acidic environment at pH of 1.5 - 3.5 for 4 – 6 hours. Then, we tested the growth of Nissle strains in the medium at pH of 2 compared to pH of 7. The results in Fig. 1 indicated that the bacterial growth was dramatically inhibited and no bacteria survived on agar plates after 5-min treatment in media at pH of 2, suggesting a safe usage of the candy with probiotics in the real world.
Fig. 1. The growth inhibition and survival rate of E. coli Nissle in LB medium and on agar plate at pH of 2. (A) The values of overnight culture were measured at OD600. The percentage of growth inhibition was calculated by dividing OD600 at pH = 7. (B) 104 bacterial cells were treated in LB broth at pH of 2 for the indicated time, followed by spreading onto the agar plate. The survival rates were ~10%, 0%, 0% for 1 min, 5 min, 30 min, respectively.
We believe it's worth creating a BioBrick-compatible pDL278 vector for application as a transforming tool in the projects among iGEM community. We got the plasmid from the lab of Dr. Yuqing Li at Sichuan University in China. Firstly, we amplified the pDL278 plasmid by PCR and assembled with the standard part of BBa_J04450 (a RFP Coding Device) in pSB1C3 to create EcoRI-XbaI and SpeI-PstI cloning sites for further BioBrick assembly.
Unfortunately, a PstI site on the pDL278 disrupts the expression of the ORF for plasmid replication, and two XbaI sites are located in the promoter region for spectinomycin resistance gene expression. We conducted 2 rounds of site-directed mutagenesis by PCR with the primers listed in Fig. 1, C. to modify these restriction enzyme sites for BioBrick assembly. The resulting Biobrick-compatible plasmid was obtained and checked by restriction enzymes (Fig. 1, B) and further confirmed by sequencing.
Unfortunately, a PstI site on the pDL278 disrupts the expression of the ORF for plasmid replication, and two XbaI sites are located in the promoter region for spectinomycin resistance gene expression. We conducted 2 rounds of site-directed mutagenesis by PCR with the primers listed in Fig. 1, C. to modify these restriction enzyme sites for BioBrick assembly. The resulting Biobrick-compatible plasmid was obtained and checked by restriction enzymes (Fig. 1, B) and further confirmed by sequencing.
POSSIBLE RISKS IN OUR EXPERIMENTS
Organisms Used and the Risk
Biosafey Level 1
✔ E. coli JM109 and E. coli DH5alpha are commercially available strains.
✔ E. coli Nissle (EcN) is a nonpathogenic bacterium isolated by Alfred Nissle in 1917. We got them from an iGEM team TAS_Taipei.
✔ Streptococcus mutans was acquired from Prof. YuQuin Li of Sichuan University in China.
All organisms we used at Biosafety Level 1 don’t pose a threat even if they escape from the lab. These organisms can't directly cause any disease to humans under general circumstances and present minimal hazard to the environment.
✔ E. coli JM109 and E. coli DH5alpha are commercially available strains.
✔ E. coli Nissle (EcN) is a nonpathogenic bacterium isolated by Alfred Nissle in 1917. We got them from an iGEM team TAS_Taipei.
✔ Streptococcus mutans was acquired from Prof. YuQuin Li of Sichuan University in China.
All organisms we used at Biosafety Level 1 don’t pose a threat even if they escape from the lab. These organisms can't directly cause any disease to humans under general circumstances and present minimal hazard to the environment.
Biosafey Level 2
✔ Streptococcus sanguinis was obtained from Prof. Ming-Shiou Jan of Chung Shan Medical University in Taiwan, who got them from ATCC, USA.
S. sanguinis is associated with human oral normal flora and could be found in biofilm in dental caries, and may be involved in infective endocarditis when existing in the bloodstream through dental procedures.
We conducted the experiments with this strain in a class II biological safety cabinet, and have safety training before all the work. We also worked on it under the supervision of experts. All the experiment will be done in Chung Shan Medical University, with a collegiate iGEM team, CSMU_Taiwan and supervised under the guidance of Prof. Jan.
✔ Streptococcus sanguinis was obtained from Prof. Ming-Shiou Jan of Chung Shan Medical University in Taiwan, who got them from ATCC, USA.
S. sanguinis is associated with human oral normal flora and could be found in biofilm in dental caries, and may be involved in infective endocarditis when existing in the bloodstream through dental procedures.
We conducted the experiments with this strain in a class II biological safety cabinet, and have safety training before all the work. We also worked on it under the supervision of experts. All the experiment will be done in Chung Shan Medical University, with a collegiate iGEM team, CSMU_Taiwan and supervised under the guidance of Prof. Jan.
Basic Parts Used and the Risk
Part: BBa_K3376000
Part name: ldhp/pSB1C3
Natural function of part: a promoter of lactate dehydrogenase gene encoding protein to produce lactate
Source: gDNA of S. mutans
Usage: used as a constitutive promoter to overexpress a protein
Species: S. mutans
Risk group: 1
Part name: ldhp/pSB1C3
Natural function of part: a promoter of lactate dehydrogenase gene encoding protein to produce lactate
Source: gDNA of S. mutans
Usage: used as a constitutive promoter to overexpress a protein
Species: S. mutans
Risk group: 1
Part: BBa_K3376003
Part name: tpxp/pSB1C3
Natural function of part: a promoter of thiol peroxidase gene encoding protein to decomposing H2O2
Source: gDNA of S. mutans
Usage: used as an inducible promoter to regulated protein expression by H2O2
Species: S. mutans
Risk group: 1
Part name: tpxp/pSB1C3
Natural function of part: a promoter of thiol peroxidase gene encoding protein to decomposing H2O2
Source: gDNA of S. mutans
Usage: used as an inducible promoter to regulated protein expression by H2O2
Species: S. mutans
Risk group: 1
Part: BBa_K3376005
Part name: AQP/pSB1C3
Natural function of part: encoding aquaporin to facilitate H2O2 bidirectional transportation
Source: IDT gene synthesis
Usage: increasing H2O2 permeation across cell membrane
Species: S. cristatus
Risk group: 2
Notes: Through the synthesis of this gene by IDT, we didn’t get in touch with this species. Besides, it's very common to find aquaporin gene in nature creatures.
Part name: AQP/pSB1C3
Natural function of part: encoding aquaporin to facilitate H2O2 bidirectional transportation
Source: IDT gene synthesis
Usage: increasing H2O2 permeation across cell membrane
Species: S. cristatus
Risk group: 2
Notes: Through the synthesis of this gene by IDT, we didn’t get in touch with this species. Besides, it's very common to find aquaporin gene in nature creatures.
Part: BBa_K3376008
Part name: SpxB/pSB1C3
Natural function of part: encoding pyruvate oxidase to generate H2O2 with antagonistic effect to S. mutans
Source: IDT gene synthesis
Usage: producing H2O2
Species: S. sanguinis
Risk group: 2
Notes: Through the synthesis of this gene by IDT, we didn’t get in touch with this species. Besides, it's very common to find pyruvate oxidase gene in nature creatures.
Part name: SpxB/pSB1C3
Natural function of part: encoding pyruvate oxidase to generate H2O2 with antagonistic effect to S. mutans
Source: IDT gene synthesis
Usage: producing H2O2
Species: S. sanguinis
Risk group: 2
Notes: Through the synthesis of this gene by IDT, we didn’t get in touch with this species. Besides, it's very common to find pyruvate oxidase gene in nature creatures.
Part: BBa_S04059 *an existing part
Part name: KatG/pSB1C3
Natural function of part: encoding catalase to decompose H2O2 to O2 and H2O for protection from oxidative stress
Source: BioBrick part
Usage: decomposing H2O2 generated by pyruvate oxidase
Species: E. coli
Risk group: 1
Part name: KatG/pSB1C3
Natural function of part: encoding catalase to decompose H2O2 to O2 and H2O for protection from oxidative stress
Source: BioBrick part
Usage: decomposing H2O2 generated by pyruvate oxidase
Species: E. coli
Risk group: 1
Risks of the manipulation
Our experiments used Bunsen burner for aseptic procedures, and we didn’t get in touch with toxic gas. In addition, the electroporation system (BTX Gemini X2) has a safety controller to prevent the accident. All alive organisms have been sterilized by bleach or autoclave. And all of the wastes were collected by a specialized agent based on the regulation of the government.
RISKS MANAGEMENT
Expert Assistance
Dr. Pei-Hong Chen oversees us when we are conducting the experiments. With a PhD degree in Molecular Biology, he is familiar with synthetic biology. He taught us with all experiment techniques. Moreover, he is also highly familiar with iGEM owing to years of experience as the instructor of the iGEM team of our school.
Guidance of Risk Management
The main regulations regarding bio-safety and bio-security, including biosafety inspections of high-containment laboratories, biosafety technical specifications and guidelines, biosafety education and training, bio-risk management post on the website of CDC Taiwan. See the guide book and the related documents here
Topics Learned
◆ Lab access and rules
◆ Responsible individuals
◆ Differences between biosafety levels
◆ Biosafety equipment (such as biosafety cabinets)
◆ Good microbial techniques (such as lab practices)
◆ Disinfection and sterilization
◆ Emergency procedures
◆ Transport rules
◆ Physical biosecurity
◆ Personnel biosecurity
◆ Dual-use and experiments of concern
◆ Data biosecurity
◆ Chemicals, fire and electrical safety
◆ Responsible individuals
◆ Differences between biosafety levels
◆ Biosafety equipment (such as biosafety cabinets)
◆ Good microbial techniques (such as lab practices)
◆ Disinfection and sterilization
◆ Emergency procedures
◆ Transport rules
◆ Physical biosecurity
◆ Personnel biosecurity
◆ Dual-use and experiments of concern
◆ Data biosecurity
◆ Chemicals, fire and electrical safety
Work Areas
◆ Open bench
◆ Biosafety cabinet
◆ Biosafety cabinet
Risk Management Tools
◆ Accident reporting (measures to record any accidents)
Personal Protective Equipment (including lab coats, gloves, eye protection, etc)
OTHER SAFETY REGULATIONS
All personnel received a tour around the lab and were informed of the rules upon entering the lab, the main rules include:
1. No running or sudden movements in the lab.
2. No eating in the lab.
3. All experiment waste must be thrown in a designated bin, which will then be put through a machine for sterilization.
4. All pipettes must be sanitized after use.
5. All equipment used must follow the user manual.
6. Experiments can only be performed with gloves and lab coats on.
7. All personnel must wash their hand upon entry into the laboratory and wear suitable clothing.
1. No running or sudden movements in the lab.
2. No eating in the lab.
3. All experiment waste must be thrown in a designated bin, which will then be put through a machine for sterilization.
4. All pipettes must be sanitized after use.
5. All equipment used must follow the user manual.
6. Experiments can only be performed with gloves and lab coats on.
7. All personnel must wash their hand upon entry into the laboratory and wear suitable clothing.
PHOTOS OF MINGDAO’S BIOLAB ABOUT SAFETY
The laminar flow clean bench
Chemical hood
Disinfection device - autoclave
