Team:Slovenia/Linker-extension standard.html

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Development of the BioBRICKS

New developed linker-extension standard
A new developed linker-extension standard is also described in detail under BBF RFC37. To improve the efficiency of cloning, we designed a NEW BioBrick standard that enables simplified and efficient linker extension between protein domains and at the same time preserve the characteristics of the most extensively used BioBrick standards. Two variations of linker-extension standard were designed. Both variations contain 5' and 3' cloning restriction sites EcoRI, PstI, NotI, XbaI and SpeI characteristic for BBa standard (Figure 1). Additionally, core restriction sites NgoMIV, AgeI, XmaI, BspEI are added. These restriction sites are used for linker extension and their positions differ among two variations of linker-extension standard. The position and the usage of these core restriction sites determine amino acid residues incorporated in the linker between protein domains (Figure 2).   Figure 1: Schematic presentation of basic elements of two linker-extension standards also named BB-NIC-II and BB-NIC-III vectors.  Figure 2: Schematic presentation of linker extension using both variations of linker-extension standards. A basic BRICK is re-cloned into the linker-extension standard using suitable restriction sites to obtain either Thr-Gly, Ser-Gly or Pro-Gly extensions.

Linker extension
Linker extension is not limited to the addition of only two amino acid residues between protein domains (Figure 3). Each round of cloning into the linker-extension standard incorporates two additional amino acid residues. The step of re-cloning could be repeated indefinitely.  Figure 3: A schematic presentation of repetitive linker extension. Similar strategy could be used to incorporate other linker amino acids. With each round of cloning the insert gains two additional amino acids. Type of linker amino acids is defined with way of cloning (restrictions of vectors).

Detailed cloning instructions
Detailed cloning instruction using the linker-extension standard A or the linker-extension standard B, BioBrick-NIC-III, are described in Figure 4 and Figure 5.  Figure 4: Schematic presentation of three different cloning strategies in multiple-cloning site of BB-NIC-II vector. Note: Each type of cloning leaves different extension of amino acids that could be used to extend scar/linker between parts after in-frame parts assembly.

 Figure 5: Schematic presentation of four different cloning strategies in multiple-cloning site of BB-NIC-III vector. Note: Each type of cloning leaves different extension of amino acids which could be used to extend scar/linker between parts after in-frame parts assembly.

Assembly strategy
Assembly strategy joining two parts/bricks is depicted in Figure 6. A detailed description is described also under BBF RFC37.  Figure 6: A schematic presentation of parts assembly. Each of two BRICKs is cut with suitable restriction enzymes and three point ligation into vector (with ccdB domain) cut with EcoRI and PstI is performed. Note: Also the standard BBa assembly strategy could be used.

Compatibility with other BBa standards
The new linker-extension standard is fully compatible with BBa and other BBa compatible standards. Each part suited for cloning into standard BioBrick vector could be cloned also into BB-NIC vectors. The part suitable for standard Biobrick vector could be cloned into BB-NIC vectors into XbaI, SpeI restriction sites with some limitations. No linker extension strategy is applicable for those parts. Transfer of parts from linker-extension standard to BBa vector could be achieved in two steps (Figure 7).  Figure 7: A schematic presentation of part transfer from BB-NIC into BBa and alike vectors. Two steps of cloning are required for directional cloning. Step 1: Ligation of part from BB-NIC into BBa both cut with XbaI/PstI. Step 2: Ligation of part from intermediate into BBa both cut with EcoRI/SpeI. }}