Team:NCTU Formosa/Safe Project Design

Safe Project Design

Choosing a non-pathogenic chassis

Bacterial strain: Escherichia coli K12

Escherichia coli K-12 are not a threat to human, animal (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, or 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 researcher, public, and environmental safety refer to Safety Q & A

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

Working with a system that can be substituted for a safer suitable system compatible with the intended work is illegal as well as immoral, even if the alternative is more difficult or expensive to acquire. In our project, safety is favored before monetary issues therefore we always attempt to locate a better host, donor, vector, insert, or system for humans, animals and the environment.

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 dispose in ‘Bio-hazardous Waste” containers


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