Team:Virginia/Background

Figure 1. Modified from Adler et al. Miniature Escherichia coli cells deficient in DNA.

What is a minicell?

Uneven cellular division in bacteria can yield a normal parent cell and an achromosomal “minicell”.

Smaller: At 400 nm, E. coli minicells are much smaller than their parental cells. [1]

Nonreplicating: Their lack of chromosomal DNA means minicells cannot replicate and cause infection, yet they still retain surface proteins and express plasmid genes.

Nonleaky: Minicells have stable, non-leaky membranes and inherit the cytosolic composition of their parent cell, maintaining protein and ion concentrations.

Targeting Potential: Since their plasma membranes are derived from the parent cell, minicells have to potential to keep membrane targeting systems put in place before their production (i.e. surface proteins like invasin or antibody coupling).

1. Make Minicell formation easily accessible to iGEM Teams

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.

2. Further Enhancing the Safety of a Minicell Chassis

To take advantage of the minicell’s innate potential as a safer biological chassis, we focused on three safety precautions to enhance suitability for the human body. First we used the lpxM strain to reduce the risks of recognition via dendritic cells and toll-like receptors (TLR-4). Secondly, we used the Ail protein and polysialic acid (PSA) to address the manifold risks posed by complement activation, a significant part of innate immunity.

• A. Reduce the Risks of TLR-4 Mediated Inflammatory Response. High concentrations of lipopolysaccharides (LPS) on bacterial cell surfaces can induce dangerous inflammatory responses that can lead to septic shock- and even death. To prevent this, we chose to work with a safer background strain. Inspired by the work of the 2007 UC Berkeley iGEM team, we worked with an lpxM deletion-insertion E. coli strain, which elicits a significantly reduced immune response. This sharp decrease in endotoxicity is due to a modification to lipid A, which acts as a hydrophobic anchor for LPS. The deletion-insertion to the lpxM gene prevents the biosynthesis of the most toxic hexa-acylated form of lipid A, instead resulting in a non-myristolated form of LPS with lower immunogenicity and a reduced capacity to activate monocyte-derived dendritic cells (as measured by a reduction in cytokines such as IL-8 and TNF-α) as well as the toll-like receptor 4 (TLR-4), which is the main signal transducing receptor for extracellular LPS. [4] Using this safer strain, we could thus mitigate the risks of a harmful inflammatory response by the innate immune system.


Figure 4. The chemical structure of lipid A as found in E. coli. Retrieved from (Raetz, Christian R. H.; Guan, Ziqiang; Ingram, Brian O.; Six, David A.; Song, Feng; Wang, Xiaoyuan; Zhao, Jinshi (2009)). "Discovery of new biosynthetic pathways: the lipid A story". Journal of Lipid Research: S103–S108.

• B. Reduce the Possibility of Complement System Activation via Ail protein and Polysialic Acid (PSA).
• The Ail protein from Yersinia enterocolitica has been proven to confer E.coli HB101 with resistance to complement killing in human serum. We aim to incorporate this protein into our minicells to prevent complement deposition, which will consequently: 1) increase minicells’ longevity in circulation and 2) reduce inflammation.
• We have also explored the possibility of encapsulating our chassis with polysialic acid (PSA), surface structures that are typically associated with the neural cell adhesion molecule (NCAM) in mammalian cells. Scavenged or synthesized de novo by certain types of bacteria, PSA reduces complement deposition as well as antibody opsonization. [4] This, too, will lengthen our minicell’s circulation time and reduce the risks of inflammation.

All three of these precautions (lpxM, Ail, PSA) will further enhance the innate safety and effectiveness of our minicells by prolonging their circulation time and preventing an adverse immune response.

[1] MacDiarmid et al (2007). Bacterially derived 400 nm particles for encapsulation and cancer cell targeting of chemotherapeutics.

[2] Ward and Lutkenhaus (1985) .Overproduction of FtsZ induces minicell formation in E. coli.

[3] Cognet, I. et al. (2003). Expression of recombinant proteins in a lipid A mutant of Escherichia coli BL21 with a strongly reduced capacity to induce dendritic cell activation and maturation.

[4] Dityatev, Alexander and Alaa El-Husseini, ed. (2006). "ch 6". Molecular Mechanisms of Synaptogenesis. New York, NY: Springer.