From 2010.igem.org

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.

1. Step: Construction of brickboxes that enable for the generation of fusion proteins

Overwiew of constructed Brickboxes . Bricks from each box are compatible and can be assembled to generate the desired 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. Due to the presence of rare codons in the sequence of PhyB and Pif3, these two genes were codon optimized and ordered from GeneArt. As the implementation of E. Lemming relies on two fusion proteins (the "anchor-light sensor" and "light sensor-che protein" fusion), two expression vectors - so called working vectors- were constructed which enable for the simultaneous expression of two fusion proteins. Working vector 1 is a derivative of pSEVA132 conferring resistance to ampicillin and replicating with ori pBB1. Working vector 2 is a derivative of pSEVA421 expressing a spectinomycin resisatnce cassette and replicating with ori RK2.

Strategy for the assembly of Fusion proteins using the generated brickboxes