Team:NCTU Formosa/Safe Project Design

Safe Project Design

Choosing a non-pathogenic chassis

Bacterial strain: Escherichia coli K-12

Escherichia coli K-12 is not a threat to humans, animals (chickens, pigs, and calves), plants or the environment, according to the 1997 Final Risk Assessment of Environmental Protection Agency (EPA). “Any concerns in terms of health considerations are mitigated by its poor ability to disseminate, colonize the colon and establish infections in a murine model” (Smith et al., 2010). In addition, the chance of the insertion gene mutating the bacterial strain to be hazardous is minimal as the inserting genetic materials have to meet the EPA’s criteria: limited in size, well characterized, free of certain nucleotide sequences, and poor mobility.

Choosing parts that will not harm humans/ animals/ plants

NameNatural FunctionAcquired ByNotes
Cell lines
H292
(Mucoepidermoid Pulmonary Carcinoma cell)		CellCompany
HCC827 (Adenocarcinoma)CellCompany
H1975 (Adenocarcinoma)CellCompany
MCF7 (Adenocarcinoma)CellCompany
MDA-MB-231(Adenocarcinoma)CellCompany
SK-BR-3 (Adenocarcinoma)CellCompany
Normal Backbone
pSB1A3Plasmid backboneKithttp://parts.igem.org/Part:pSB1A3
pSB1K3Plasmid backboneKithttp://parts.igem.org/Part:pSB1K3
pSB1C3Plasmid backboneKithttp://parts.igem.org/Part:pSB1C3
Expression Backbone
pSB6A1Plasmid backboneKithttp://parts.igem.org/Part:pSB6A1

Inserts:

NameNatural FunctionAcquired ByNotes
Promoters
J23101Consitutive promoterKithttp://parts.igem.org/Part:BBa_J23101
J23110Consitutive promoterKithttp://parts.igem.org/Part:BBa_J23110
R0010Induced promoterKithttp://parts.igem.org/Part:BBa_R0010
Ribosome Binding Site (RBS)
B0034Strong; efficiency 1.0Kithttp://parts.igem.org/Part:BBa_B0034
B0030Strong; efficiency 0.91Kithttp://parts.igem.org/Part:BBa_B0030
Fluorescent Reporters
E0040Green Fluorescent ProteinKithttp://parts.igem.org/Part:BBa_E0040
E1010Red Fluorescent ProteinKithttp://parts.igem.org/Part:BBa_E1010
K592100Blue Fluorescent ProteinKithttp://parts.igem.org/Part:BBa_K592100
Chromoprotein
K592009amilCPKithttp://parts.igem.org/Part:BBa_K592009
Transmembrane Protein
Lpp-OmpA fusion proteinTransmembrane protein in E.coliPaper
FadLTransmembrane protein in E.coliPaper
Single-chain variable fragment (scFv) antibodies
scFv of Anti-EGFRPart of AntibodyDrug bank
scFv of Anti-VEGFPart of AntibodyDrug bank
scFv of Anti-HER2Part of AntibodyPaper
Gold Binding Polypeptide (GBP)
Gold Binding PolypeptidePaper
Terminator
J61048TerminatorKithttp://parts.igem.org/Part:BBa_J61048

Risks involving the use of Genetically Engineered Organisms (GEOs)

Our GEOs are composed of standard promoters, ribosomal binding sites, terminators and fluorescent reporters, and have no known risks. The function and known homologs are classified by BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and researched (mainly via PubMed). For more information on the researcher, public, and environmental safety refer to Safety Q & A

Substituting safer materials for dangerous materials in a proof-of-concept experiment

It is illegal and immoral to work with a system if it could be substituted for a safer suitable system that is also compatible with the intended work even if the alternative is more difficult or expensive to acquire. In our project, safety is favored before monetary issues. Therefore an attempt is always made to locate a better host, donor, vector, insert, or system for humans, animals and the environment in order to ensure that the system is the safest it can be.

Gel Electrophoresis: Ethidium bromide → Invitrogen SYBR Safe

Since Ethidium bromide is a very toxic agent and acts as a mutagen (intercalates double stranded DNA), we have changed our nucleic acid staining molecules to Invitrogen SYBR Safe, which is significantly less toxic and better for the environment (may have direct disposal into the wastewater drainage systems). In case EtBr or Invitrogen SYBR Safe affects DNA biological processes, these substances are added in minimal concentration to the agar solution before it solidifies.

*Use of gloves is mandatory and must be disposed in ‘Bio-hazardous Waste” containers
Gel Electrophoresis: Transilluminator → Digital Gel Imaging Systems

We usually use UV radiation to visualize the stained DNA in agar gels. However, long exposure to UV light is harmful, carcinogenic, and destroys vitamin A. Therefore, we replaced UV transilluminator with digital gel imaging systems to avoid close UV contact. This reduces the necessity of special safety lenses and other special equipment that were used to block the harmful effects of ultraviolet. UV is also used in laminar flow cabinets for sterilization and decontamination purposes. For safety precautions, we have limited the sterilization process to 30 minutes with no other personnel in the laboratory.

Safety: Paraformaldehyde → Sodium Azide → Tetracycline, Ampicillin, Sulfonamide

At first paraformaldehyde was considered a good choice because the substance was known for its fixation properties. However, the level of toxicity led many to question our disposal system. In addition, paraformaldehyde did not fulfill our project needs. We wished to have constant fluorescent intensity with decreasing or constant cell count but the use of paraformaldehyde hindered this ability as it lacked constant fluorescent intensity.

Next we considered sodium azide. This substance is a strong respiratory toxin which inhibits the activity of cytochrome oxidase and is similar to a bacteriostatic agent. However, this chemical is also highly toxic to mammals. Therefore, we did not experiment with sodium azide.

Sodium azide led us to explore all kinds of bacteriostatic agents. Tetracycline, ampicillin, and sulfonamide are just some of the bacteriostatic agents that we researched. Tetracycline attaches to 30s ribosomal subunit and inhibits protein synthesis causing bacteria cell count to slowly decrease. Ampicillin inhibits additional formation of cell membrane and sulfonamide obstructs DNA replication, therefore in both cases cell splitting becomes impossible.

Including an "induced lethality" or "kill-switch" device

Safety is one of the most important aspects of iGEM. When biosafety is not considered properly, many life forms can be destroyed. To start, we decided to experiment with paraformaldehyde. The renowned fixation properties led us to believe that our E.coli can be considered nonliving while it continues to function. However, the results of our experiments were not favorable. In addition, the toxicity of paraformaldehyde itself raised questions. The search for other alternatives led us to bacteriostatic agents.

There are numerous varieties of bacteriostatic agents, such as glycylcycline, macrolide, oxazolidinone, and tetracycline. In the preceding experiments, tetracycline, ampicillin, and sulfonamide were researched. These substances represent different types of inhibition to stop cell replication. Tetracycline attaches to 30s ribosomal subunit and inhibits protein synthesis causing bacteria cell count to slowly decrease. Ampicillin inhibits additional formation of cell membrane and sulfonamide obstructs DNA replication. Therefore, in both cases cell splitting becomes impossible.



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