Team:Virginia/Background

Uneven cellular division in bacteria can yield a normal parent cell and an achromosomal “minicell”. At 400 nm, E. coli minicells are much smaller than their parental cells. Their lack of chromosomal DNA means they cannot replicate and cause infection, yet they still retain and express plasmid genes. Additionally, minicells have stable, non-leaky membranes and inherit the cytosolic composition of their parent cell, maintaining protein and ion concentrations. Since their plasma membranes are derived from the parent cell, minicells keep any targeting systems put in place before their production. Perhaps the best complements to minicell’s modularity, membrane-bound antibodies serve as highly-specific and effective ways to direct minicells to a target. These unique qualities make minicells a viable and safer alternative to comparable chassis such as liposomes and standard E. coli.

Minicells have great potential in a synthetic biology context. We aim to create a standard BioBrick that induces minicell formation in E. coli cells and to design additional safeguards to make our chassis as safe as possible. Research has shown that the overproduction of the tubulin-homolog FtsZ leads to minicell formation. Accordingly, we designed a simple BioBrick to contain the ftsz gene under the control of an IPTG-inducible promoter. This allows us to tune FtsZ production to achieve optimal minicell production. We are developing a model that relates the amount of IPTG added to the final concentration of minicells that are produced and purified.

Despite minicells being unable to replicate, their safety for medicinal use remains an utmost concern. To address this, we seek to reduce or even eliminate the threat of an inflammatory response induced through by presence of LPS and complement deposition. The Ail protein from Yersinia pestis is especially promising in that regard, as it confers resistance to the human innate immune response. We have also explored the possibility of encapsulating our chassis with polysialic acid, mimicking the mammalian structures typically associated with the neural cell adhesion molecule (NCAM). This too reduces complement deposition as well as antibody opsonization. Both Ail and polysialic acid can be produced in concert with our original FtsZ BioBrick to create safer minicells in a desired strain of E. coli. For our purposes, we are using an lpxM mutant strain that renders the conserved Lipid A portion of LPS less toxic through a reduction in acyl-group modifications.

Minicells lack the ability to replicate and therefore carry virtually no risk of infection, which is especially important for the young, the elderly, or the immunocompromised. The Ail, polysialic acid and IpxM mutant modifications we will implement will further enhance their innate safety, lowering risks associated with an adverse immune response. By combining multiple safety elements with an inherent ease of production and modularity, we hope to showcase minicells’ potential as chassis for therapeutic use and consumption.