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Team:ETHZ Basel/Biology/Cloning
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As we plan to generate several fusion proteins with different linkers, we decided to use the cloning strategy BBF RFC28: A method for combinatorial multi-part assembly based on the Type IIs restriction enzyme AarI (http://dspace.mit.edu/handle/1721.1/46721). The advantage of this strategy is that we can clone up to 3 different inserts into one vector simultaneously in a 96 well format. | As we plan to generate several fusion proteins with different linkers, we decided to use the cloning strategy BBF RFC28: A method for combinatorial multi-part assembly based on the Type IIs restriction enzyme AarI (http://dspace.mit.edu/handle/1721.1/46721). The advantage of this strategy is that we can clone up to 3 different inserts into one vector simultaneously in a 96 well format. | ||
| - | == generation of | + | == Generation of different Brickboxes that enable for the generation of a variety of chemotaxis fusion proteins== |
| - | + | Parts were generated by PCR using primers specified in the BBF RFC28 manual and subcloned into the storage vector pSEVA132 (Victor de Lorenzo's lab, KanR, pBBR1 ori) by blunt end ligation. pSEVA132 allows for blue white screening, making the generation of the brickbox parts very efficient. Generated parts were verified by AarI digest and sequencing using primer M13r. | |
| - | + | Due to the presence of rare codons in the sequence of PhyB and Pif3, these two genes were codon optimized and ordered from GeneArt. | |
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| - | Due to the presence of rare codons in the sequence of PhyB and Pif3, these two genes | + | |
== Submitted parts == | == Submitted parts == | ||
Revision as of 08:18, 14 October 2010
BioBricks
As we plan to generate several fusion proteins with different linkers, we decided to use the cloning strategy BBF RFC28: A method for combinatorial multi-part assembly based on the Type IIs restriction enzyme AarI (http://dspace.mit.edu/handle/1721.1/46721). The advantage of this strategy is that we can clone up to 3 different inserts into one vector simultaneously in a 96 well format.
Generation of different Brickboxes that enable for the generation of a variety of chemotaxis fusion proteins
Parts were generated by PCR using primers specified in the BBF RFC28 manual and subcloned into the storage vector pSEVA132 (Victor de Lorenzo's lab, KanR, pBBR1 ori) by blunt end ligation. pSEVA132 allows for blue white screening, making the generation of the brickbox parts very efficient. Generated parts were verified by AarI digest and sequencing using primer M13r. Due to the presence of rare codons in the sequence of PhyB and Pif3, these two genes were codon optimized and ordered from GeneArt.
Submitted parts
| <groupparts>iGEM010 ETHZ_Basel</groupparts> |
