Team:Slovenia/Linker-extension standard.html

New developed linker-extension standard

A new developed linker-extension standard is also described in detail under [http://dspace.mit.edu/bitstream/handle/1721.1/46705/BBFRFC37.pdf?sequence=1 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.