BBa_K4703025/BBa_K4703026: HML-MazF/EcoRI

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We have connected the cis-acting elements HML-I(BBa_K4703006) and HML-E(BBa_K4703007) on either side of the toxin gene MazF(BBa_K4703015) or EcoRI(BBa_K4703008). HML-I and HML-E recruit the Sir2/Sir3/Sir4 complex, which can form and maintain heterochromatin^[1]. Without altering the DNA sequence, we suppress the expression of the toxin gene through epigenetic modifications, allowing cells carrying the toxin gene to survive.

Figure 1. The principle of HML-MazF/HML-EcoRI

BBa_K4703031: dCas9-sir2 fusion protein expression cassette

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The dCas9-sir2 fusion protein(BBa_K4703016) is transcribed under the control of the TEF1 promoter(BBa_K4703017) from Saccharomyces cerevisiae. Within the dCas9-sir2 fusion protein, sir2 possesses histone deacetylase activity, and dCas9 can specifically bind to the target site. Therefore, this fusion protein can specifically induce histone deacetylation at the target DNA sequence, forming heterochromatin, and thereby inhibiting the expression of the target gene. The terminator used is the CYC1 Terminator (BBa_K3112005).

Figure 2. The principle of dCas9-sir2 fusion protein

Other Parts we used

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Name	Part Type	Short Description
BBa_K4703000	Protein coding sequences	dCas9
BBa_K4703001	Terminator	S.pyogenes terminator
BBa_K4703002	Protein coding sequences	dCas9-dam fusion protein
BBa_K4703003	Regulatory	dnaAP2 promoter
BBa_K4703004	Regulatory	mioC promoter
BBa_K4703005	Protein coding sequences	GFP
BBa_K4703006	DNA	HML-I
BBa_K4703007	DNA	HML-E
BBa_K4703008	Protein coding sequences	EcoRI-NLS
BBa_K4703009	RNA	sgRNA-ccdB-1
BBa_K4703010	RNA	sgRNA-ccdB-2
BBa_K4703011	RNA	sgRNA-ccdB-3
BBa_K4703012	RNA	sgRNA-mioC-1
BBa_K4703013	RNA	sgRNA-mioC-2
BBa_K4703014	Regulatory	TetR-tet promoter
BBa_K4703015	Protein coding sequences	MazF-NLS
BBa_K4703016	Protein coding sequences	dCas9-sir2 fusion protein
BBa_K4703017	Regulatory	TEF1 promoter
BBa_K4703018	Composite	mioC-RFP-dam DNA methylation system
BBa_K4703019	Reporters	dnaAP2-RFP
BBa_K4703020	Reporters	mioC-RFP
BBa_K4703021	Composite	trc-dCas9
BBa_K4703022	Composite	mioC-ccdB
BBa_K4703023	Reporters	tetR-tet-GFP
BBa_K4703024	Composite	mioC-ccdB-dam kill system
BBa_K4703025	Composite	HML-MazF
BBa_K4703026	Composite	HML-EcoRI
BBa_K4703027	Composite	trc-dCas9-dam
BBa_K4703028	Composite	pTDH3-MazF-CYC1
BBa_K4703029	Composite	pTDH3-EcoRI-CYC1
BBa_K4703030	Reporters	T7lac-RFP
BBa_K4703031	Composite	dCas9-sir2 fusion protein expression cassette
BBa_K4703032	Reporters	CHO-sgGFP
BBa_K4703033	Reporters	CYC1-sgGFP
BBa_K4703034	RNA	sgRNA-scaffold
BBa_K4703035	Plasmid	p15A-dCas9
BBa_K4703036	Plasmid	p15A-dCas9-Dam
BBa_K3121004	Regulatory	trc promoter
BBa_K3189008	Regulatory	LacI
BBa_K2142004	Protein coding sequences	Dam DNA methylase
BBa_K247000	Protein coding sequences	mRFP1
BBa_R0187	Regulatory	T7 promoter (lacI repressible)
BBa_K4244056	Protein coding sequences	ccdB
BBa_S03368	Regulatory	pCI
BBa_B0010	Terminator	S.pyogenes terminator
BBa_K530008	Regulatory	pTDH3 promoter
BBa_K3112005	Terminator	CYC1 Terminator
BBa_M50049	Terminator	T7 terminator