DNA Anchor/Terminator

Anchoring System

The current method for Byte production (ie: USER^TM) necessitated a particular anchoring system. Longer sticky ends were also desired to increase the efficiency of recircularization. These factors led to the development of a USER^TM-based anchoring system. An anchoring piece, constructed of two annealed oligomers, is bound to the streptavidin-coated bead via a 5' biotin modification and provides a sticky 3' overhang complementary to an A end. Once the desired number of Bytes is added, a terminator (again, two annealed oligomers) is annealed and ligated to the available end of the final brick (in this case, a B end). The entire construct is then treated with USER^TM enzyme mix. The resulting end product from the digestion of uracil contained within the anchor, anneals to the terminator overhang and can be ligated to form a circular product. The ligation also yields a complete SceI site that can be used to linearize the construct for recombination into the E. coli genome. See Figure 1.

A vital component of the BioBytes method is the use of a biotinylated DNA anchor piece in order to allow unidirectional assembly of the Bytes on paramagnetic beads. The anchor itself has three vital components: A 5’ biotinylation, a 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. Our team has considered a number anchor systems, each with their own set of advantages and disadvantages. The three main anchor systems we investigated are summarized below. See Table 1.

Table 1: Overview of three different anchoring systems that were considered for BioBytes.

Figure 1: pAB and pBA multiple cloning sites with highlighted primers prA1/B1u and prA2/B2u annealing regions.