Team:ETHZ Basel/Biology/Cloning

From 2010.igem.org

(Difference between revisions)
(→Generation of Brickboxes that enable for the generation of fusion proteins)
(→Generation of Brickboxes that enable for the generation of fusion proteins)
Line 8: Line 8:
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.  
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.
Due to the presence of rare codons in the sequence of PhyB and Pif3, these two genes were codon optimized and ordered from GeneArt.
-
[[Image:picture6.png|thumb|400px|'''Schematical overview of the devices and change upon light pulse induction.''' LSP refers to light switch protein, AP to anchor protein, anchor to the plasmid anchor and Che to the attacked protein of the chemotaxis pathway.]]
+
[[Image:picture6.png|thumb|500px|'''Overwiew of constructed Brickboxes .''' Bricks from each box are compatible and can be assembled to generate the desired fusion proteins.]]
== Strategy for the assembly of Fusion proteins ==
== Strategy for the assembly of Fusion proteins ==

Revision as of 09:15, 14 October 2010

Cloning strategy for the construction of our 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.

Generation of Brickboxes that enable for the generation of 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.

Overwiew of constructed Brickboxes . Bricks from each box are compatible and can be assembled to generate the desired fusion proteins.

Strategy for the assembly of Fusion proteins

<groupparts>iGEM010 ETHZ_Basel</groupparts>
Retrieved from "http://2010.igem.org/Team:ETHZ_Basel/Biology/Cloning"