Team:Freiburg Cloning Strategy

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_cloning strategy

Step 1	Vector digestion: EcoRI + PstI	Insert digestion: EcoRI + PstI
	BBa-J52017	_CMV-promotor
Step 2	Vector digestion: AgeI+SpeI	Insert digestion: NgoMIV+SpeI
	pMA-BBFR _ SPLIT-Linker	C-YFP
	pMA-BBFR _ SPLIT-Linker	C-CFP
Step 3	Vector digestion: AgeI+SpeI	Insert digestion: NgoMIV+SpeI
	pMA-BBFR _egfR-Tm	_ N-β-Lactamase
		_ C-β-Lactamase
		_ SPLIT-Linker_ C-YFP
		_ N-YFP
		_ SPLIT-Linker_ C-CFP
		_ N-CFP
		_ BB058 (Luciferase)
		_ BB057 (Luciferase)
Step 4	Vector digestion: AgeI+SpeI	Insert digestion: NgoMIV+SpeI
	pMA-BBFR _SP	_scFv-anti-NIP
	pMA-BBFR _SP	_ Lipocalin
Step 5	Vector digestion: AgeI+SpeI	Insert digestion: NgoMIV+SpeI
	pMA-BBFR _SP_ scFv-anti-NIP and pMA-BBFR-+SP_ Lipocalin	_GGGS-linker (produced by Klenow fill in)
Step 6	Vector digestion: AgeI+SpeI	Insert digestion: NgoMIV+SpeI
	pMA-BBFR _SP_ scFv-anti-NIP _ GGGS-Li and pMA-BBFR _ SP_ Lipocalin _ GGGS-Li	_ egfR-Tm _ N-β-Lactamase
		_ egfR-Tm _ C-β-Lactamase
		_ egfR-Tm _ SPLIT-Linker_ C-YFP
		_ egfR-Tm _ N-YFP
		_ egfR-Tm _ SPLIT-Linker_ C-CFP
		_ egfR-Tm _ N-CFP
		_ egfR-Tm _ BB058 (Luciferase)
		_ egfR-Tm _ BB057 (Luciferase)
Step 7	Vector digestion: SpeI + PstI	Insert digestion: XbaI + PstI
	BBa-J52017_CMV	_SP_ scFv-anti-NIP_GGGS-Li_egfR-Tm_N-β-Lactamase
		_ SP_ scFv-anti-NI _GGGS-Li_ egfR-Tm_C-β-Lactamase
		_ SP_ scFv-anti-NIP_GGGS-Li_ egfR-Tm_SPLIT-Linker_C-YFP
		_ SP_ scFv-anti-NIP_GGGS-Li_ egfR-Tm_N-YFP
		_ SP_ scFv-anti-NIP_GGGS-Li_ egfR-Tm_SPLIT-Linker_C-CFP
		_ SP_ scFv-anti-NIP_GGGS-Li_ egfR-Tm_N-CFP
		_ SP_ scFv-anti-NIP_GGGS-Li _ egfR-Tm_BB058 (Luciferase)
		_ SP_ scFv-anti-NIP_GGGS-Li _ egfR-Tm_BB057 (Luciferase)
		_ SP_ Lipocalin _GGGS-Li_ egfR-Tm_N-β-Lactamase
		_ SP_ Lipocalin _GGGS-Li_ egfR-Tm_C-β-Lactamase
		_ SP_ Lipocalin _GGGS-Li_ egfR-Tm_SPLIT-Linker_ C-YFP
		_ SP_ Lipocalin _GGGS-Li_ egfR-Tm_N-YFP
		_ SP_ Lipocalin _GGGS-Li_ egfR-Tm_SPLIT-Linker_ C-CFP
		_ SP_ Lipocalin _GGGS-Li_ egfR-Tm_N-CFP
		_ SP_ Lipocalin _GGGS-Li__ egfR-Tm _ BB058 (Luciferase)
		_ SP_ Lipocalin _GGGS-Li__ egfR-Tm _ BB057 (Luciferase)

Figure 1 . A shows the Biobrick part BBa_J52017. The transfectionvektor for eukaryotic cell systems has an ampicillin- and a kanamycin resistance cassette. The multiple cloning site contains the Biobrick standard restriction sites EcoRI, NotI, XbaI, SpeI, NotI, PstI followed by an eukaryotic terminator sequence. B The CMV-promotor fragment was obtained by PCR with the Biobrick BBa-J52038 template. C The PCR product was cloned into the transfectionvector by EcoRI and PstI to get a final eukaryotic transfection-system. D To test the efficiency of expression a gene-fragment coding for the yellow fluorescent protein was put into the vector behind CMV-Promotor.

Figure 2 . A figure 2 A gives an overview about the cloning constructs. The N-terminal signal-peptide ensures protein transport to the cytoplasmamembrane. Lipocalin and the scFv-ani-NIP are the extracytoplasmatic parts of the construct to mediate signaltransduction into the cell. The GGGS-Linker keeps a distance to the transmembraneregion to overcome surface structures of the cell and to avoid a total inflexibility. Split fluorophor linker is only necessary for the C-terminal Split parts of Cerulean-CFP and Split-Venus-YFP. The Split enzymes β-Lactamese and Luciferase and the split-fluorophors CFP and YFP are the cytoplasmatic parts of the constructs. If there is a clustering of this synthetic receptor-system caused by the corresponding binding parts of Lipocalin and scFv-anti-NIP the split parts come together to create a functional protein, which allows a detection. B The different constructions described in figure 2.A were cloned into the transfectionvector system by using the restriction sites XbaI and PstI to ensure a functional ATG-start codon which is part of the XbaI recognition-sequence in the iGEM-prefix