Chromosomal Assembly

Once sizable chunks of synthetic chromosome have been constructed, it becomes necessary to find a manner by which to assemble the pieces together and insert them into the cell, while simultaneously replacing corresponding regions of the original host chromosome. One approach would be to fabricate the entire construct in vitro in the form of a bacterial artificial chromosome (BAC), insert the BAC, and then inactivate the host chromosome. However, we have adopted the approach of piecing together the chromosome in vivo by recombining synthetic sections into the original host chromosome. This provides a step-wise means of testing the functionality of smaller gene subsets rather than attempting to find errors in an entire minimal chromosome.

In Vivo Construction

This method requires a technique known as Lambda Red recombination. Synthetic sections produced via the BioBytes method can be transformed through electroporation as linear fragments. Once a fragment is in a cell, the Red recombination genes direct the section to a double crossover event at regions on the chromosome homologous to regions flanking the ends of the synthetic section. This results in the replacement of a large portion of the original chromosome with a synthetic construct. For a fully synthetic genome, this process can be repeated until only the original Ori remains.

Advantages of Recombination Over Building BAC's

• For one, it can be very difficult to transform a 400 kb construct into E. coli as a single piece. An in vivo approach allows for the transformation of a number of much smaller constructs.
• By building the synthetic chromosome onto the original chromosome, there is no need for a complicated method of “rebooting” the cell. Transforming the entire new chromosome would require a way to smoothly inactivate or destroy the host chromosome without killing the cell in the process. In vivo construction allows for a smooth transition from the original to the artificial cell.
• The step wise integration of synthetic material into the host genome also allows for greater ability to test the functionality of subsets of the artificial genome. If the cells die upon integration of a synthetic fragment, then there may be a problem with the new construct, or the construct has displaced an essential gene without replacing it.