Team:Freiburg Cloning Strategy

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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-anti-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-Lactamase 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

[[METHODS]]
The cloning was started with a preparative digestion of the DNA-Plasmids. To clone fusion parts the vector constructs were digested with AgeI and PstI to open the Biobrick suffix. The inserts were digested with NgoMIV and PstI. For cloning into the transfection-vector the enzymes SpeI and PstI were used for vector and XbaI, PstI for insert to keep up the ATG-start codon in the XbaI restriction site of the biobrick suffix. All restriction-enzymes were ordered from New England Biolabs. After digestion the DNA-fragments were separated on a 1% agarose gel. The DNA-band of interest was isolated and purified with the QIAGEN QIAquick Gel Extraction Kit. For the ligation a 3 molar excess of the insert was put together with the vector-fragment and ligated with a Quick ligase (New England Biolabs). After half an our at room temperature the DNA was transformed to chemical competent E.coli strain XL1 cells, plated on 2YT-agar-plates and incubated at 37°C over night. After picking clones and growing in 5ml LB-medium, the plasmid DNA was isolated by QIAGEN QIAprep Spin Miniprep Kit. A test digestion was prepared with about 0,5µg Plasmid DNA and NotI restriction enzyme to isolate the fusion-protein from the vector and to control if the expected bands were obtained. After a positive result the clones were sent to GATC-Biotech for sequencing. The GGGS-Linker was produced by Klenow -fill-in-PCR. Two primers were designed align to each other at 60°C and filled to a complete dobble-strand by Klenow Polymerase fragment.
Digestion Protocol

 - about 2µg Plasmid-Prep in 20µl 

 - 2 µl NEB Buffer 10x

 - 1 µl NEB enzyme 1 (NgoMIV, AgeI, XbaI, EcoRI)

 - 1 µl NEB enzyme 2 (PstI, SpeI)

 - 0,2µl BSA 100x

Ligation

 - 10µl volume of vector and insert DNA (about  50ng vector-DNA)

 - 1 µl DNA Quick Ligase (New England Biolabs)

 - 10 µl Quick Ligase Buffer

Analytic digestion

 - about 0,5 µg Plasmid-DNA in 5µl

 - 5µl H2O

 - 0,5 µl NotI

 - 1µl NEB-Buffer

 - 0,1 µl BSA

Transformation

   Competent cells (100µl) werde defrosted on ice

   10µl of the ligation was added

   DNA and cells werde mixed softly 

   Incubation on ice for 20-30 min

   Heat shock at 42°C for 40 sek 

   cells were cooled down on ice for 5-10 min

   900µl sterile 2YT Medium was added

   Incubation at 37°C for 60-70 min (shaker)

   cells werde plated on 2YT-agar-plates with antibiotics

Klenow fill in reaction

 - 25pmol forward primer
 - 25pmol reverse primer
 - 0,5 µl Klenow-fragment without exonuclease activity (Fermentas)
 - 2µl Klenow Buffer
 - 1µl dNTPs
 - Add H¬¬2O to a volume of 20µl
   program: 94°C for 3min, cool down to 37°C, adition of klenow enzyme, 37°C for 1 hour