Parts

Implemented Genetic Parts

This section presents a table organized according to each part of the genetic construct. It includes the name of each component, its function within the genetic circuit, its location within each Construct (we built three constructs designed to detect mercury, as detailed in the design section), the database code for each element, and the category to which the individual parts belong.

Constitutive Promoters:

Name	Function	Construct	Code	iGEM parts
Pc429-1	Medium-strength constitutive promoter	2, 3	BBa_K5257018	New Basic Part
PcJ109	Weak constitutive promoter	1	BBa_J23109	iGEM parts
PLlacO-1	IPTG-inducible promoter, regulated by the LacI repressor (lac operon)	3	BBa_R0011	iGEM parts

Inducible Promoters:

Name	Function	Construct	Code	iGEM parts
PTn21	Mercury-inducible promoter derived from Tn21	1	BBa_K5257017	New Improved Part
PmerT	Mercury-inducible promoter from Tn501	2	BBa_K5257015	New Improved Part
PM16	Mutated low-expression Hg²⁺-inducible promoter from Tn501	2	BBa_K5257016	New Improved Part

RBS (Ribosome Binding Sites):

Name	Function	Construct	Code	iGEM parts
RBS34	Very strong ribosome binding site	1,2,3	BBa_B0034	iGEM parts
RBS32	Weak ribosome binding site	1,2,3	BBa_B0032	iGEM parts
RBS31	Medium-strength ribosome binding site	2,3	BBa_B0031	iGEM parts
RBS30	Strong ribosome binding site	2,3	BBa_B0030	iGEM parts

Terminators:

Name	Function	Construct	Code	iGEM parts
TerL3S2P21	Very strong synthetic terminator	1,2,3	BBa_K2675031	iGEM parts
TerL3S2P55	Strong synthetic terminator	1,2	BBa_K2598008	iGEM parts
TerL3S2P22	Strong synthetic terminator	2	BBa_K3320000	iGEM parts
TerL3S3P56	Strong synthetic terminator	3	BBa_K5257002	New Basic Part

Sensors:

Name	Function	Construct	Code	iGEM parts
merRm4-1	aTF repressor-activator sensitive to inorganic and organic mercury	1	BBa_K5257010	New Basic Part
merRtn501	aTF repressor-activator sensitive to inorganic mercury	2	BBa_K5257011	iGEM parts

Reporters:

Name	Function	Construct	Code	iGEM parts
sfGFPmChartreuse	Optimized green fluorescent reporter	2	BBa_K5257004	New Basic Part
mRFP_Magenta	Magenta fluorescent chromoprotein	2	BBa_K5257000	New Basic Part

Coding Genes:

Name	Function	Construct	Code	iGEM parts
OmpTLOT	Extracellular surface anchor motif, includes lpp signal peptide	3	BBa_K5257009	New Basic Part
gdh	Glucose dehydrogenase, provides NADPH using D-glucose	3	BBa_K5257008	New Basic Part
gomerB	Alkylmercury lyase enzyme, catalyzes the cleavage of the organic group from mercury	3	BBa_K5257006	New Basic Part
merA_2D	Mercuric reductase enzyme, catalyzes Hg²⁺ to Hg⁰ conversion. Requires NADPH. Thermostable and efficient from Pseudomonadota bacterium	3	BBa_K5257007	New Basic Part New Improved Part
TEV	TEV protease with S219V mutation, prevents auto-cleavage	2	BBa_K3450016	iGEM parts
tetO	Binding site for aTF repressor TetR	1	BBa_K2066502	iGEM parts

Auxiliary Peptides: Fusion, Anchoring, Signaling, Degradation

Name	Function	Construct	Code	iGEM parts
G4Slinker	Linker peptide between Gly and Ser protein domains	1,2,3	BBa_K2549053	iGEM parts
tevsite-Fdegron	TEV protease cutting site specific ENLYFQ/- with N-tag for protein degradation, highly efficient FLFVQ degron	1,3	BBa_K5257021	New Basic Part
ctxBSP	Signal peptide from ctxB protein, detected by the Type II secretion system's SEC pathway	3	BBa_K5257014	New Basic Part
DogCatcher	Tagging peptide, post-translational protein fusion system	3	BBa_K5257013	New Basic Part
DogTag	Fusion tag, post-translational protein fusion system	3	BBa_K5257012	New Basic Part
10xN-HisTag	His N-terminal label for protein purification	1,2	BBa_K1162009	iGEM parts
10xC-HisTag	His C-terminal label for protein purification	3	BBa_K844000	iGEM parts

New Composite Parts

Name	Function	Construct	Code	iGEM parts
NOT(TetR,TEV)-mRFP1magenta	Magenta reporter controlled by NOT logic gate	1	BBa_K5257025	BBa_K2066502 BBa_K2066502 BBa_B0034 BBa_K1162009 BBa_K5257021 BBa_K2549053 BBa_K5257000
N-10xHistag TEVprotease	Easily purified TEV protease	2	BBa_K5257001	BBa_K1162009 BBa_K2549053 BBa_K3450016
LOT-DogCatcher	Anchor motif with a fusion peptide	3	BBa_K5257019	BBa_K5257009 BBa_K2549053 BBa_K5257013
gdh-DogTag	gdh enzyme with a fusion peptide, fuses with LOT-DogCatcher	3	BBa_K5257020	BBa_K5257008 BBa_K2549053 BBa_K5257012 BBa_K844000
DogTag-MerA_2D	MerA_2D enzyme with a fusion peptide, fuses with LOT-DogCatcher	3	BBa_K5257022	BBa_K5257014 BBa_K5257012 BBa_K2549053 BBa_K5257021 BBa_K5257007
LOT-MerA-GDH	Enzymes on the cell surface, a system for volatilization of Hg2+ without the need for NADPH.	3	BBa_K5257023	BBa_R0011 BBa_B0034 BBa_K2549053 BBa_B0030 BBa_K5257022 BBa_B0031 BBa_K5257020 BBa_K2675031

WHITE LIST. The host of this biosensor is E. coli DH5α which is in the lowest risk category (Biosafety Level 1, BSL-1) according to microbiological safety guidelines. This means that it can be safely handled in basic laboratory settings without significant risk to human or animal health. We are also not releasing this bacteria outside the laboratory, in case a horizontal transfer of these resistance genes would not generate any ill effects on other bacteria as no method of eliminating bacteria is based on mercury. These resistant bacteria will convert all methylmercury into inorganic mercury (less dangerous species) and inorganic mercury into elemental mercury (even less dangerous) so the action of this bacteria if accidentally released is remedial.