Using our devised assembly protocol we first tested the system with alternating AB and BA Bytes. To keep things simple, all AB Bytes were hemF (900bp), and all BA Bytes were rplB (822bp). We started with the A anchor, then added AB hemF, followed by BA rplB. We alternated AB Bytes with BA Bytes until we had added 8 Bytes total (4 of each). Next, the construct was digested off the bead via USER^TM mix. The construct was then run on a gel (Figure 1). We were able to assemble seven Bytes in a day.

Figure 1.

As you can see, the assembly process is not very efficient yet. To see what we've done to increase efficiency of ligation, check out the optimization.