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Team:Potsdam Bioware/Project/Details
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First mdnA was deleted by excising using the restriction enzymes SfoI and AatII. The next step comprised the introduction of a mdnA-geneIII-fusion gene. Therefore gene III was amplified from pak100blaKDIR and mdnA from pARW089 by PCR. The primers were designed to enable the introduction of iGEM and other restriction sites required for further cloning steps. The purified PCR product gene III was digested by NgoMIV and AatII whereas the PCR product mdnA was digested by SfoI and AgeI. Subsequent the three fragment ligation of mdnA and gene III into the digested vector has been conducted. Thus a mdnA-gene III fusion part according to RFC25 was generated whereby AgeI and NgoMIV overhangs are compatible and placed in frame with the protein sequence. The ligation of AgeI and NgoMIV overhangs resulted in a scar coding for the threonine and glycine. Because the introduction of restriction sites before mdnA leaded to a great distance between ribosome binding site and mdnA a second RBS was inserted among SfoI and XbaI recognition sites to ensure a sufficiently expression rate of the mdnA-geneIII-fusion gene. The myc sequence located between mdnA and gene III allows the detection of the expression of the mdnA-geneIII-fusion protein on the surface of the phage using western blot or ELISA. | First mdnA was deleted by excising using the restriction enzymes SfoI and AatII. The next step comprised the introduction of a mdnA-geneIII-fusion gene. Therefore gene III was amplified from pak100blaKDIR and mdnA from pARW089 by PCR. The primers were designed to enable the introduction of iGEM and other restriction sites required for further cloning steps. The purified PCR product gene III was digested by NgoMIV and AatII whereas the PCR product mdnA was digested by SfoI and AgeI. Subsequent the three fragment ligation of mdnA and gene III into the digested vector has been conducted. Thus a mdnA-gene III fusion part according to RFC25 was generated whereby AgeI and NgoMIV overhangs are compatible and placed in frame with the protein sequence. The ligation of AgeI and NgoMIV overhangs resulted in a scar coding for the threonine and glycine. Because the introduction of restriction sites before mdnA leaded to a great distance between ribosome binding site and mdnA a second RBS was inserted among SfoI and XbaI recognition sites to ensure a sufficiently expression rate of the mdnA-geneIII-fusion gene. The myc sequence located between mdnA and gene III allows the detection of the expression of the mdnA-geneIII-fusion protein on the surface of the phage using western blot or ELISA. | ||
| - | [[File:UP cloning strategy.png|400px]] | + | [[File:UP cloning strategy.png|500px]] [[File:UP pPDV089.png|400px]] |
=== In Vivo Selection === | === In Vivo Selection === | ||
Revision as of 11:16, 20 September 2011
Details
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Microviridin
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Phage Display
Cloning
The phage display vector pPDV089 was derived from the plasmid pARW089 which carries the whole mdn-cluster. This plasmid contains a plasmid origin of replication and additionally a f1 ori which enables the packaging of single strand DNA into phages. For selective amplification ampcillin and kanamycin resistence genes are included. To create the phagemid pPDV089 standard cloning procedure were performed.
First mdnA was deleted by excising using the restriction enzymes SfoI and AatII. The next step comprised the introduction of a mdnA-geneIII-fusion gene. Therefore gene III was amplified from pak100blaKDIR and mdnA from pARW089 by PCR. The primers were designed to enable the introduction of iGEM and other restriction sites required for further cloning steps. The purified PCR product gene III was digested by NgoMIV and AatII whereas the PCR product mdnA was digested by SfoI and AgeI. Subsequent the three fragment ligation of mdnA and gene III into the digested vector has been conducted. Thus a mdnA-gene III fusion part according to RFC25 was generated whereby AgeI and NgoMIV overhangs are compatible and placed in frame with the protein sequence. The ligation of AgeI and NgoMIV overhangs resulted in a scar coding for the threonine and glycine. Because the introduction of restriction sites before mdnA leaded to a great distance between ribosome binding site and mdnA a second RBS was inserted among SfoI and XbaI recognition sites to ensure a sufficiently expression rate of the mdnA-geneIII-fusion gene. The myc sequence located between mdnA and gene III allows the detection of the expression of the mdnA-geneIII-fusion protein on the surface of the phage using western blot or ELISA.
In Vivo Selection
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Modelling
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