Overview

Initial Strategy

We are developing a streptavidin based system for the rapid and flexible assembly of great lengths of DNA. To serve as an anchor for the growing DNA chain, biotinylated single-stranded molecules of DNA are bound to streptavidin-coated paramagnetic beads. These beads allow a rapid centrifugation-free method for the exchange of wash/DNA solutions. Once the biotinylated DNA molecules are bound to the streptavidin-coated beads, a 17 base universal initiator is annealed to the single-stranded anchor. This creates a 4 base overhang that is compatible with a Not I digested DNA fragment. See Figure 1A.

Figure 1A: Diagrammatic representation of biotinylated anchor bound to streptavidin as well as initiator region and subsequently added bricks

The 1st DNA “brick” to be added to the growing chain is cultivated from standard plasmids (either pAB or pBA) by restriction digest with Not I, Nb.BbvCI and Nb.BtsI. This yields a fragment with an overhang compatible with that of the initiator. The fragment is thus annealed and ligated to the initiator overhang. The other terminus of the DNA brick is comprised of a 12 base overhang known as either “A” (if taken from pBA) or “B” (if taken from pAB). See Figure 1B.

Figure 1B: pAB and pBA multiple cloning sites with highlighted Not I and Nb.BbvCI/Nb.BtsI overhangs

More bricks are added to the tethered chain after their removal from standard plasmids pAB and pBA. Removal of a DNA brick from pAB requires restriction digest using BspQI, Nb.BbvCI and Nb.BtsI. This yields a brick with a 12 base A overhang 5’ of the brick cassette and a 12 base B overhang 3’ of the brick cassette. See Figure 2A.

Figure 2A: pAB multiple cloning sites with highlighted A and B 12 base overhangs

Removal of a DNA brick from pBA requires restriction digest using BsmBI, Nb.BbvCI and Nb.BtsI. This yields a brick with a 12 base B overhang 5’ of the brick cassette and a 12 base A overhang 3’ of the brick cassette. See Figure 2B. AB and BA bricks may also be PCR'd up using universal primers prA1/prB1 and prA2/prB2 respectively. This reduces the enzymes required to create the 12 base overhang to Nb.BbvCI and Nb.BtsI. PCR is the preferred method as it excludes the use of BspQI and BsmBI (which function optimally at 55/50 deg. Celsius). Regardless of creation method, these bricks are assembled sequentially onto tethered fragment via annealing of 12bp A and B regions followed by ligation.

Figure 2B: pBA multiple cloning sites with highlighted B and A 12 base overhangs

The tethered chain of DNA is terminated by addition of a cap (either complementary to the A overhang or the B overhang). The ligation of the Cap to the A or B overhang creates a sticky end compatible with Not I digested DNA. The construct is digested with Not I. This releases the 1st brick from the initiator/biotinylated DNA complex while creating a sticky end that will anneal to that of the Cap. The circularized DNA is then ligated a final time and transformed into E. coli using standard techniques. See Figure 3.

Figure 3: Ligated terminators Cap (A) and Cap (B) annealed to Not I overhang created by digestion. DNA fragment is then ligated to recircularize before transformation into E. coli

Current Strategy

Brick Creation

The use uracil-containing primers and USER(TM) enzyme mix provides an alternative (and more effective) method for creating 12 base sticky ends. The primers anneal to the A and B regions respectively, as well as ~5bp 3' into the cassette (to increase melting temperature). Bricks cloned into pAB and pBA can be PCR'd up with these universal uracil primers prA1u/prB1u, prA2u/prB2u) and treated with USER(TM) mix. The uracil DNA glycosylase (UDG) present will cleave the uracil base and endonuclease VIII will subsequently cleave the sugar-phosphate backbone at the apyrimidinic, creating single stranded regions which can be purified away using PCR purification spin columns. See Figure 4.

Figure 4: pAB and pBA multiple cloning sites with highlighted primers prA1/B1u and prA2/B2u annealing regions

Anchoring System

The new and improved method for brick production (ie: USER^TM) necessitated a change in 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. When the desired number of bricks 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 5.

Figure 5: Showing anchor and terminator fragments and effect of USER^TM treatment. I SceI site and A ends are highlighted