Personal safety

Before we started all our work in the lab, all the team members had to receive training courses in which we learned the importance of safety rules, including that eating and drinking are not allowed in the lab, and never operating experiments alone. Also, we should wear personal protection equipment, such as lab coats, gloves, and goggles, as operating experiments.

Laboratory safety

The experiments in our project can be conducted in a biosafety level 1 laboratory. In addition, the operation of bacteria and volatile chemicals should be performed in the biosafety cabinets and fume hoods, respectively. Besides, we also learn the use and precautions of various machinery in the laboratory, and understand what hazards might be encountered in the laboratory and how to deal with them when they occur. Furthermore, we must immediately report to our advisor or safety center if there is any accident or equipment damage.

Environmental safety-E.coli

Three strains of E. coli strand are applied in our experiment as follows, DH5-alpha, BL21 (DE3) and DH10-beta.

• The DH5-alpha E.coli is used to amplify the vectors containing biobricks for protein expression, in vivo rolling circle replication, and in vitro rolling circle amplification.
• The BL21(DE3) E.coli is used to express mGL (Green fluorescence protein) and PBSII-Zif268 fusion protein, which are applied to CTC detection and tetrahedron anchorage, respectively.
• The DH10-beta E.coli is used to express ssDNA by in vivo rolling circle replication.

These three strains belong to the biosafety level 1, which are low risk to human beings. According to the biosafety rules in experiment operation, all the biowastes will be sterilized by autoclaving, and the environment will be sterilized by bleaching or decontaminated by 75% EtOH.

Dimethylformamide (DMF)

DMF serves as aprotic solvent in SN2 reaction, which plays an essential role in DNA tetrahedron connecting with folic acid. DMF liquid and vapor pose hazardous threats to human health. DMF vapor could cause acute toxicity when inhaltion, and DMF liquid can lead to serious damage to eyes, skin, and respiratory system when directly contacted. The overexposure of DMF liquid or vapor even leads to decreased fertility and certain organs damage. To avoid the above negative impacts from DMF liquid and vapor, we wore gloves and carefully operated DMF in the biosafety cabinet .

Ethidium bromide (EtBr)

EtBr is applied in agarose gel electrophoresis to verify whether DNA tetrahedron was successfully assembled or not. EtBr is a potential mutagen that will intercalate double-stranded DNA and RNA. Inhaltion of excessive EtBr is even deadly. We set up a separate EtBr-only zone for EtBr-related experiments and always equipped with gloves when conducting experiments in this zone. The used EtBr will be destroyed by sunlight exposure for two days.

PMSF (phenylmethylsulfonyl fluoride)

PMSF is used to inhibit the serine protease function to prevent protein degradation. PMSF is a corrosive and irritant liquid to skin. To prevent hazards caused by PMSF, we wear gloves during PMSF-related experiments to refrain from any possible contact with PMSF.

DCC(N,N'-Dicyclohexylcarbodiimide)

DCC is a kind of Carbodiimide, which reacts with folic acid to form an intermediate product in the DNA tetrahedron-folic acid linkage experiment. DCC is highly irritating to eyes and probably causes acute toxicity and allergic reaction to skin while direct contact. We operate DCC in the chemical fume hood with gloves and goggles to avoid the above negative impacts.

Chloroform

Chloroform is used to adhere Polymethyl methacrylate boards. Chloroform liquid causes skin irritation when direct contact with skin, and would pose substantial damage to liver and kidney when prolonged or repeated exposure. The team members are required to wear gloves and always operate chloroform in the chemical fume hood to prevent negative health impacts.

Ether

Ether is applied to wash away the residual reactant and DMF in NHS-folic acid conjugation. Both liquid and vapor of ether are extremely flammable and could probably cause drowsiness and dizziness when inhalation. We operate ether in the chemical fume hood with masks and prevent using sparking and flammable tools with ether such as alcohol burner , to avoid the above potential hazard.