Part:BBa_K187074

A vital component of the BioBytes method is the use of a biotinylated DNA Anchor in order to allow unidirectional assembly of the Bytes on paramagnetic beads by sequestering the 5' ends of Bytes, leaving only the 3' ends available to bind incomding Bytes.

This part can be annealed to the anchor reverse strand (BBa_K187422) to produce an anchor that has an B end. The end can be annealed to the B' end produced from a gene in plasmid pBA.

The anchor itself has three vital components: A 5’ biotinylation, a double stranded DNA (dsDNA) portion that incorporates a release mechanism in order to liberate the construct from the beads, and A or B overhangs to allow Bytes to bind to the Anchor.

The BioBytes anchor system utilizes a 5’-biotin which anchors the construct to beads by binding non-covalently, but with great strength, to the covalently linked streptavidin on the surface of the paramagnetic beads. There is also a 5’-15 nucleotide spacer region of ssDNA which facilitates more efficient binding of the Anchor to the bead as the binding pocket of streptavidin is deep and thus a highly flexible ssDNA linker is recommended to allow the biotin to effectively bind into this deep pocket. A 21 bp double stranded portion of the Anchor contains the I-SceI recognition sequence, which when digested with I-SceI produces 4 base overhangs, but also includes four deoxyuracil residues. These uracils are excised by New England Biolab’s USERTM system to generate single nucleotide gaps in the top strand. USERTM digestion thus effectively destroys the Anchor and produces a 21 base 3’ overhang which becomes important for recircularization of the construct. Finally the Anchor contains the A or B 3’ overhangs complementary to those of the Bytes, allowing their binding to the Anchor.