Random Homology-Aided Transformation for Genome and Plasmid Streamlining

Synthetic biologists aim to achieve predictable and controllable genetic devices, yet they have to struggle against the massive complexity already present within the simplest living cell. In this context, any synthetically implemented process will unavoidably experience some level of interference that may alter its desired outcome. One way to approach this problem is to remove all non-essential components from a genome, a feat that so far has shown both promise and limitations. Although up to 16% genome reductions have been achieved in E. coli without any significant loss in cellular fitness (Posfai et al., 2006), most attempts to achieve broad scale genome reductions have negatively impacted cellular growth. Dispensable genes have been found to function in stress responses and allow for efficient usage of resources to build new cells (Karcagi et al., 2016). For these reasons, it is extremely difficult to determine which genomic regions can be removed without compromising a cell’s full viability. 

We aimed to develop a method capable for generating combinatorial libraries of deletion variants that retain full cellular viability. Taking advantage of the high transformability of the naturally competent bacterium Acinetobacter baylyi ADP1, we tested the possibility of using fragmented genomic DNA (gDNA) religated to a selective marker to template homology-aided random genome deletions. By designing the marker cassette with Type IIS terminal restriction enzyme sites facing outwards, a mutant’s digested genomic DNA contains fragment with the marker gene attached to 16-bp sequences flanking where it was integrated into the genome. These sequences serve to map out the precise site of deletion/integration event. We named this combinatorial methodology for generating pools of random deletion mutants random Homology-Aided Transformation (rHAT). Using rHAT we were able to retrieve fit and viable mutants with deletions averaging ~5kb (smallest 1832bp and largest 9227bp; 20 isolates assayed in total), although a surprising number of the retrieved transformants (>50%) had undergone genome re-arrangements that apparently lead to duplications in the genome while still generating a local deletion in one copy of an amplified region. Determining which genomic regions remain robust through normal or induced mutational processes can greatly benefit genome engineering projects and synthetic biologist, e.g., by helping to map out both dispensable and unstable genomic regions which would be unsuitable for genome engineering.

Furthermore, the demonstration that ADP1 can be efficiently transformed via rHAT may prove valuable for evolutionary genome reduction experiments. For example, we expect that evolving a population of cells with the continual input of randomly religated gDNA will likely accumulate deletions over evolutionary time, thereby serving to evolve fit variants with reduced genomes or deletions which may then be combined to make a minimal ADP1 genome. Future experiments will also test rHAT as a combinatorial approach to generate pools of minimized plasmids in ADP1 hosts; smaller plasmids are typically easier to transform into a cell. Overall, our preliminary results using rHAT, open the door to the use and development of this method as a tool to achieve simpler, more efficient, and more reliable synthetic biology.