Introduction

Significant Composite Part

To ensure system biosafety, we developed a blue light-inducible suicide circuit. While the pDawn promoter is widely used for blue light-mediated gene expression, its three-level cascade architecture results in suboptimal efficiency and non-negligible basal leakage. To address these limitations, we sought, through literature research, an alternative blue light induction strategy that is more efficient and less architecturally complex, thereby introducing an innovative improvement over existing designs.

We identified the LOV protein, whose Jα helix undergoes conformational unwinding upon blue light irradiation. This property has been exploited to create the LOVdeg tag, in which a protease-recruiting short peptide is inserted into a conformationally dynamic region of the LOV domain. By fusing the LOVdeg tag to a target protein, blue light can trigger its specific degradation.

For controlled expression of the suicide pathway, a repressible promoter is required. Among classic systems such as the lac and tet operons, we selected the tet operon to avoid potential interference from endogenous LacI expression in Escherichia coli. The Tet repressor (TetR), a homodimeric protein, was fused to the LOVdeg tag. Upon blue light illumination, TetR bound to the operator is degraded, thereby rendering the tet operon light-inducible—representing an innovative transition from chemical induction to optogenetic control.

Downstream of the inducible promoter, we employed MazF, a toxin from the toxin–antitoxin system, which induces cell death through RNA degradation.

Part Collection

Basic Parts

	Type	Part number	Name	Brief introductions
1	Basic	BBa_25TUX7U5	echM	an enoyl-CoA hydratase with high sequence identity to known E. coli enoyl-CoA hydratases
2	Basic	BBa_25VYPHLI	gspM	contains a complete general stress protein family GsiB domain
3	Basic	BBa_25RFSX0M	the LOVdeg tag	an optogenetic protein regulatory tool, enables blue light-controlled degradation of target proteins
4	Basic	BBa_255YSB76	tetR-LOVdeg-6xHis	The Tet repressor (TetR), a dimer, is tagged with LOVdeg. Under blue light, TetR bound to the operator is degraded, rendering the Tet operon light-inducible—an innovative shift from chemical induction to optogenetics.
5	Basic	BBa_2518L5MR	ag43-2xFLAG tag	a protein specifically mediates cell aggregation, adhesion, and biofilm development
6	Basic	BBa_25VLMD2F	6xHis-csgA-GSGGSG-RYRYRYR	the protein linker, which connects arginine - rich short peptides to curli protein, induces spore sedimentation
7	Basic	BBa_257N6ODM	OmpA+10xHis	a highly conserved outer membrane protein which facilitates the translocation of extracellular matrix (ECM) components to maintain biofilm structural stability.
8	Basic	BBa_25UW1D69	tet operator	A Tet repressor-operator system is suitbale for BBa_255YSB76.

Composite Parts

	Type	Part number	Name	Brief introductions
9	Composite	BBa_25QGYS4W	Ag43-OmpA expression pathway（T7）	biofilm enhancing pathway
10	Composite	BBa_25OKVA0Z	EchM-GspM expression pathway（T7）	salt-tolerant genes, gspM and echM to arm our biofilm with the necessary adaptability against inhospitable conditions
11	Composite	BBa_25XCX0NU	RBS + intimin-Nb3+sfGFP expression pathway（T7）	expression of surface-displayed nanobodies (Nb3) and sfGFP for observation
12	Composite	BBa_25AJPRS2	RBS + intimin-Ag3+mCherry expression pathway（T7）	expression of surface-displayed corresponding antigens (Ag3) and mCherry for observation
13	Composite	BBa_25BG1LK9	TetR-LOVdeg+sfGFP expression pathway	blue light induced quantification system
14	Composite	BBa_25IEE64L	TetR-LOVdeg+mazF expression pathway	blue light induced suicide system