This year´s main project is the attempt to create an "artificial receptor-system", featuring extra- and intracellular modules as well as suitable transmembrane regions.
The intracellular domaine of our receptor-device is build by halves of split reporter-proteins that can reassemble and will then produce readable output, e. g. fluorescence.
Each one of these protein-halves is connected to its extracellular domaine by a single-span transmembrane-helix.
The extracellular or detecting domaine consists of a protein or peptide with the ability to bind a certain molecule.
Now, if a system with two matching receptors is presented these molecules in a strict, pairwise spatial arrangement, the receptor-devices are brought together,
the split reporter-protein reassembles inside the cell and the output can be detected.
We employ so-called "Origami-DNA" to create the exactly defined molecule-patterns that are needed to activate our receptors.
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Step 1
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Vector
digestion: EcoRI + PstI
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Insert
digestion: EcoRI + PstI
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BBa-J52017
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_CMV-promotor
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Step
2
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Vector
digestion: AgeI+SpeI
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Insert
digestion: NgoMIV+SpeI
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pMA-BBFR
_ SPLIT-Linker
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C-YFP
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C-CFP
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Step
3
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Vector
digestion: AgeI+SpeI
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Insert
digestion: NgoMIV+SpeI
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pMA-BBFR
_egfR-Tm
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_ N-β-Lactamase
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_ C-β-Lactamase
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_ SPLIT-Linker_ C-YFP
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_ N-YFP
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_ SPLIT-Linker_ C-CFP
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_ N-CFP
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_ BB058 (Luciferase)
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_ BB057 (Luciferase)
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Step
4
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Vector
digestion: AgeI+SpeI
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Insert
digestion: NgoMIV+SpeI
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pMA-BBFR
_SP
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_scFv-anti-NIP
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_ Lipocalin
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Step
5
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Vector
digestion: AgeI+SpeI
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Insert
digestion: NgoMIV+SpeI
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pMA-BBFR
_SP_ scFv-anti-NIP
and
pMA-BBFR-+SP_ Lipocalin
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_GGGS-linker (produced by Klenow fill in)
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Step
6
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Vector
digestion: AgeI+SpeI
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Insert
digestion: NgoMIV+SpeI
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pMA-BBFR
_SP_ scFv-anti-NIP _ GGGS-Li
and
pMA-BBFR
_ SP_ Lipocalin _
GGGS-Li
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_
egfR-Tm _ N-β-Lactamase
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_
egfR-Tm _ C-β-Lactamase
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_
egfR-Tm _ SPLIT-Linker_ C-YFP
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_
egfR-Tm _ N-YFP
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_
egfR-Tm _ SPLIT-Linker_ C-CFP
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_
egfR-Tm _ N-CFP
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_
egfR-Tm _ BB058 (Luciferase)
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_
egfR-Tm _ BB057 (Luciferase)
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Step
7
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Vector
digestion:
SpeI + PstI
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Insert
digestion: XbaI + PstI
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BBa-J52017_CMV
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_SP_ scFv-anti-NIP_GGGS-Li_egfR-Tm_N-β-Lactamase
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_ SP_ scFv-anti-NI _GGGS-Li_ egfR-Tm_C-β-Lactamase
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_ SP_ scFv-anti-NIP_GGGS-Li_
egfR-Tm_SPLIT-Linker_C-YFP
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_ SP_ scFv-anti-NIP_GGGS-Li_ egfR-Tm_N-YFP
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_ SP_ scFv-anti-NIP_GGGS-Li_
egfR-Tm_SPLIT-Linker_C-CFP
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_ SP_ scFv-anti-NIP_GGGS-Li_ egfR-Tm_N-CFP
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_ SP_ scFv-anti-NIP_GGGS-Li _ egfR-Tm_BB058 (Luciferase)
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_ SP_ scFv-anti-NIP_GGGS-Li _ egfR-Tm_BB057 (Luciferase)
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_ SP_ Lipocalin _GGGS-Li_ egfR-Tm_N-β-Lactamase
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_ SP_ Lipocalin _GGGS-Li_ egfR-Tm_C-β-Lactamase
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_ SP_ Lipocalin _GGGS-Li_
egfR-Tm_SPLIT-Linker_ C-YFP
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_ SP_ Lipocalin _GGGS-Li_
egfR-Tm_N-YFP
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_ SP_ Lipocalin _GGGS-Li_
egfR-Tm_SPLIT-Linker_ C-CFP
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_ SP_ Lipocalin _GGGS-Li_
egfR-Tm_N-CFP
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_ SP_ Lipocalin _GGGS-Li__ egfR-Tm _ BB058 (Luciferase)
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_ SP_ Lipocalin _GGGS-Li__ egfR-Tm _ BB057 (Luciferase)
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"
