Team:Alberta/Project/assemblyoverviewold

BioBytes constitutes the University of Alberta’s contribution to iGEM 2009. It is a next-generation gene assembly system with the singular potential to accelerate the field toward the grand vision of the artificial cell. Unlike BioBricks, genes produced in the BioByte format can be assembled rapidly in vitro, in any desired order, with great precision and yield. With cycle times approaching 20 minutes for the addition of each new gene, BioByte assembly rates exceed their BioBrick counterpart by 200-fold. This level of improvement immediately opens the door towards the synthesis of simple chromosomes that can be tested and optimized at unprecedented speed. Finally, the BioByte assembly system requires a fraction of the equipment found in a conventional gene lab. This advantage, combined with a "child-could-do-it" simplicity, greatly extends its utility from the high school classroom to the workbench of the bio-process engineer.

A BioByte is a piece of DNA that encodes a specific type of cellular function or instruction. Each end of the DNA is distinct from the other so that they so that they can only be joined in a head-to-tail fashion as shown below.

Figure 1.

Once correctly paired, Bytes are irreversibly linked together by a special enzyme called DNA ligase. It is the strength and accuracy of the head-to-tail interaction that accounts for BioByte’s superior yield, precision and speed relative to its BioBrick predecessor.

The hand-in-glove nature of the BioByte interaction illustrates how the orientation of each Byte is determined, but how can this approach be used to control the order by which Bytes are assembled?

Figure 2.

The problem of determining Byte order is shown in Figure 2. Here, the free ends on the existing two-byte chain are such that the incoming third brick can be added randomly to either end, resulting in the order [1-2-3] or [3-1-2]. BioBytes has overcome this problem by using a third type of end that can only be linked to an inert magnetic microsphere (Figure 3). By anchoring the first byte to the microsphere via this new end, only one end, its free end, is available for interaction. The chain therefore is constrained to grow in only one direction: away from its anchor. The microsphere design also fulfills another important function. Microspheres stick to magnets. Anchored chains can therefore be moved out of one reaction mixture into another containing new Byte molecules needed for the next round of addition. Unlinked Bytes are left behind.

Figure 3.

At this stage, the problem of Byte order and chain fidelity is not entirely solved. The method described above cannot exclude the possibility that multiple copies of a particular Byte become incorporated at a given step as shown in Figure 4 below.

Figure 4.

The problem arises because each Byte that is incorporated into the chain does not enter the reaction mixture as a single molecule, but as a population of identical molecules that are as likely to interact with each other as the anchored chain (Figure 5).

Figure 5.

BioBytes solves the problem by constructing each Byte in two alternative forms: an “A” form and a “B” form. Each form has two incompatible ends. Therefore neither form can be linked to itself (Figure 6). The ends of each form are compatible with each other, allowing for the alternating order of A and B forms in head-to-tail orientation. Adding Bytes to the growing chain by alternating the A an B forms assures that only one copy of each is added at each step.

Figure 6.

Upon completion of the desired product, chains are released from the microspheres by a chemical cleavage event that separates the anchor brick from its bound end. Full length constructs can then be introduced into living cells.

With its BioBytes approach, the Alberta team has recently demonstrated the accurate construction of chains composed of 10 Bytes over the course of 4 hours with no obvious limit to the final length that can be achieved. A key advantage to this approach is that it can be multiplexed for the production of multiple chains simultaneously that can be linked together as SuperBytes by the same method to produce artificial chromosomes of unprecedented length.