Safety Regulations

All personnels 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 suitbale clothings.

---

Photos of the LAB:

The BIOLAB at Mingdao High School.

Eating and Drinking are not allowed in the Lab.

The Laminar Flow Clean Bench in the BIOLAB, MDHS.

Warning Signs are posted within the LAB.

The Emergency Eyewash and Autoclave device in the BIOLAB, MDHS.

Our Goal

High concentration of carbon dioxide in an enclosed space can make people feel sleepy and even dizzy. While driving, drivers may fall asleep. Accidents might occur. To solve this problem, we plan to use the cyanobacteria with high photosynthetic efficiency to reduce the concentration of CO2. However, the solubility of CO2 in water is low, so we modified Bacillus spp. to produce human carbonic anhydrase (CA), which can increase the solubility of CO2 in water. Furthermore, to extend the application, we produced CYP2E1 from rabbits, which is used in xenobiotic metabolism, to eliminate benzene and chloroform. To sum up, we built up an efficient microalgae purification system to improve indoor air quality using natural enzymes such as carbonic anhydrase and xenobiotic metabolic enzymes.

Possible Risks

Organisms Used

E. coli DH5alpha, Bacillus subtilis 168, Synechococcus elongatus PCC 7942, Spirulina spp.

Risks of Selected Organisms

We use two kinds of algae: Synechococcus elongatus PCC 7942 (non-toxic lab strain, recognized as a safe organism and exists in nature) and Spirulina spp (edible food supplement), and they will not pose a threat even if it escapes from the lab.

Organisms Used

• Escherichia coli: E.coli DH5 alpha
• Bacillus subtilis

Risks of Organisms Used

Bacillus subtilis 168 (Generally Regarded As Safe) and E.coli strain are recognized within the Biosafety Level 1 by the US Public Health Service Guidelines and pose a subtle risk to humans, and are not known to consistently cause disease in immunocompetent adult humans, and present a minimal potential hazard to laboratory personnel and the environment.

Parts Used and the Risk

Basic parts information

Part: BBa_K2932000

Name: Carbonic anhydrase (KEGG: Synpcc7942_1388) / pSB1C3
Natural function of part: an enzyme catalyzing the interconversion between carbon dioxide/water and bicarbonate/hydrogen ions
Source: gDNA of Synechococcus elongatus PCC7942
Usage: to increase CO2 dissolving rate
Species: Synechococcus elongatus PCC7942
Risk Group: 1
Risk to humans or environment: No Disease: No

Part: BBa_K2932001

Name: Carbonic anhydrase (KEGG: Synpcc7942_1447)/ pSB1C3
Natural function of part: an enzyme catalyzing the interconversion between carbon dioxide/water and bicarbonate/hydrogen ions
Source: gDNA of Synechococcus elongatus PCC7942
Usage: to increase CO2 dissolving rate
Species: Synechococcus elongatus PCC7942
Risk Group: 1
Risk to humans or environment: No Disease: No

Part: BBa_K2932002

Name: Carbonic anhydrase (human CAII) / pSB1C3
Natural function of part: an enzyme catalyzing the interconversion between carbon dioxide/water and bicarbonate/hydrogen ions
Source: a plasmid got from Addgene
Usage: to increase CO2 dissolving rate
Species: Homo sapiens (human)
Risk Group: No (Animals and plants have no risk group)
Risk to humans or environment: No Disease: No

Part: BBa_K2932003

Name: CYP2E1 / pSB1C3
Natural function of part: an enzyme catalyzing the xenobiotic metabolism including benzene, chloroform, etc.
Source: DNA synthesis by Twist Bioscience company
Usage: to eliminate Volatile organic compounds (VOCs)
Species: Oryctolagus cuniculus (rabbit)
Risk Group: No (Animals and plants have no risk group)
Disease: No

Composite parts information

To express enzymes of CA or CYP2E1 in Bacillus subtilis 168, we make the following composite parts using a Bacillus promoter PliaI, which is controlled by the LiaRS antibiotic-inducible two-component system.

Part: BBa_K2932004

PliaI promoter (BBa_K823001) + RBS (BBa_B0034) + CA1388 (BBa_K2932000) + Terminator (BBa_B0015)

Part: BBa_K2932005

PliaI promoter (BBa_K823001) + RBS (BBa_B0034) + CA1447 (BBa_K2932001) + Terminator (BBa_B0015)

Part: BBa_K2932006

PliaI promoter (BBa_K823001) + RBS (BBa_B0034) + hCA (BBa_K2932002) + Terminator (BBa_B0015)

Part: BBa_K2932007

PliaI promoter (BBa_K823001) + RBS (BBa_B0034) + CYP2E1 (BBa_K2932003) + Terminator (BBa_B0015)

Experiment Design

We set up a system containing algae, enzyme and culture media. We then prepared the culture media with standard media of BG-11 or baking soda, liquid fertilizer, leaf fertilizer to adjust the ratio of composition and change the illuminance, temperature to find out the appropriate environment to optimize algal growth. We prepare raw lysates from Bacillus subtilis 168 producing CA or CYP2E1 by breaking down by 0.1 um glass beads, we didn’t further purify the specific enzymes and just used the total lysates as active biocatalysts. To test CO2 elimination by CA, we use indoor CO2 (500~1000 ppm) or CO2 tanks (2000 ppm and 100%) to provide the gases and analyze CO2 concentration with infrared CO2 sensor in a chemical hood. As to test chloroform, we asked NCTU-Formosa team to help in a specialized chemistry and engineering lab.

Risks of the Experiment

Our experiment only compares the growth of algae under different conditions and the solubility of carbon dioxide, so it will not pose any danger. For chloroform analysis, we collaborated with experts at the NCTU-Formosa team in a specialized lab.

Project Usage

• A consumer product that can be bought by all people
• A small and enclosed device

Risks of Usage

The device we designed only contains two organisms and two kinds of enzyme currently: algae (edible Spirulina spp and lab strain Synechococcus elongatus PCC 7942) and enzymes (human carbonic anhydrase and rabbit CYP2E1). Even if the device is unfortunately broken, the former won't pose a threat to the environment. While the latter will be degraded naturally. Since the materials we use won't have any negative effect, safety, security or ethical risks won’t be considered.

Risks Management

Biosafety Level 1

Expert Assistance

Dr.Pei-Hong Chen will oversee us when we are doing the experiments. He has a PhD degree in Molecular Biology and is familiar with synthetic biology experiments. In addition, he is highly familiar with iGEM due to his years of experience as our school's iGEM coach.

Guidnace of Risk Management

We underwent trainings on lab skills and safety by our instructor, Dr, Pei-Hong Chen, before working in the biotech laboratory. We always strictly followed the guidance of biological laboratory regulation at Taiwan given by Academia Sinica, the national academy of Taiwan. See the guide book here.

Topics Learned

• Lab access and rules (including appropriate clothing, eating and drinking, etc.
• Responsible individuals (such as lab or departmental specialist or institutional biosafety officer)
• 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

Risk Management Tools

• Accident reporting (measures to record any accidents)
• Personal Protective Equipment (including lab coats, gloves, eye protection, etc)