Team:Virginia

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Preliminary Description:

Minicells

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 renders them unable to replicate and cause infection, yet they still retain and express plasmid genes (Mugridge). Additionally, minicells have stable, non-leaky membranes and inherit the cytosolic composition of their parent cell, maintaining the same protein and ion concentrations (Frazer). Since their plasma membranes are derived from the parent cell, minicells also retain any targeting systems put in place before their production. Perhaps the best complements to minicells’ modularity, membrane-bound antibodies enable highly-specific and effective targeting of minicells to specific cells (MacDiarmid). These unique qualities make minicells a viable and safer alternative to comparable chassis such as liposomes and standard E. coli.

Minicells hold 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 (Ward). Accordingly, we designed a simple BioBrick to contain the ftsz gene under the control of an IPTG-inducible promoter. This allows us to adjust FtsZ production to achieve optimal minicell production. We are simultaneously developing a quantitative model that relates the amount of IPTG added to the final concentration of minicells that are produced and purified.

Despite minicells’ inability 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 the presence of lipopolysaccharide (LPS) and via complement deposition. The Ail protein from Yersinia pestis is especially promising in that regard, as it confers resistance to the human innate immune response (Kolodziejek). 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). Sialylation would further reduce complement deposition, as well as antibody opsonization. In other words, 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. As an additional safety precaution, we also decided to use the lpxM mutant strain, which has a reduction in the acyl-group modifications of the conserved Lipid A portion of its LPS. Studies have shown that this variant can reduce E-selectin stimulation and TNF-α production by up to 10,000-fold (Anisimov). Combined, these three safeguards could hence significantly slash the immunogenicity and risks associated with a minicell chassis.

In conclusion, 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 associated health risks. By combining multiple safety elements with an inherent ease of production and modularity, we hope to showcase minicells' potential as a chassis for widespread therapeutic use and consumption.

Sources

Anisimov, A., Shalkhutdinova, R., Pan, L., Feodorova, V., Savostina, E., Bystrova, O., Lindner, B., & Mokrievich, A. (2007). Effect of deletion of the lpxm gene on virulence and vaccine potential of yersinia pestis in mice. Journal of Medical Microbiology, 56(4), 443-53. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/17374882?dopt=AbstractPlus

Frazer, A. & Curtiss, 3rd R. (1975). Production, properties and utility of bacterial minicells. Current Topics in Microbiology and Immunology. 69, 1–84.

Kolodziejek, A., Schnider, D., Rohde, H., Wojtowicz, A., Bohach, G., Minnich, S., & Hovde, C. (2010). Outer membrane protein x (ail) contributes to yersinia pestis virulence in pneumonic plague and its activity is dependent on the lipopolysaccharide core length. Infectious Immunology, 78(12), 5233-43. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/20837715

MacDiarmid, J. A., Mugridge, N. B., Weiss, J. C., et al. (2007). Bacterially derived 400 nm particles for encapsulation and cancer cells targeting of chemotherapeutics. Cancer Cell, 11(5), 431-45.

@@ Line 22: / Line 22: @@
 </p>
-<p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Minicells hold great potential in a synthetic biology context. We aim to create a standard BioBrick that induces minicell formation in <i>E. coli</i> 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 (Ward). Accordingly, we designed a simple BioBrick to contain the ftsz gene under the control of an IPTG-inducible promoter. This allows us to adjust FtsZ production to achieve optimal minicell production. We are simultaneously developing a quantitative model that relates the amount of IPTG added to the final concentration of minicells that are produced and purified.
+<p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Minicells hold great potential in a synthetic biology context. We aim to create a standard BioBrick that induces minicell formation in <i>E. coli</i> 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 (Ward). Accordingly, we designed a simple BioBrick to contain the <i>ftsz</i> gene under the control of an IPTG-inducible promoter. This allows us to adjust FtsZ production to achieve optimal minicell production. We are simultaneously developing a quantitative model that relates the amount of IPTG added to the final concentration of minicells that are produced and purified.
 </p>
-<p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Despite minicells’ inability 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 the presence of lipopolysaccharide (LPS) and via complement deposition. The Ail protein from Yersinia pestis is especially promising in that regard, as it confers resistance to the human innate immune response (Kolodziejek). 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). Sialylation would further reduce complement deposition, as well as antibody opsonization. In other words, both Ail and polysialic acid can be produced in concert with our original FtsZ BioBrick to create safer minicells in a desired strain of <i>E. coli</i>. As an additional safety precaution, we also decided to use the lpxM mutant strain, which has a reduction in the acyl-group modifications of the conserved Lipid A portion of its LPS. Studies have shown that this variant can reduce E-selectin stimulation and TNF-α production by up to 10,000-fold (Anisimov). Combined, these three safeguards could hence significantly slash the immunogenicity and risks associated with a minicell chassis.
+<p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Despite minicells’ inability 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 the presence of lipopolysaccharide (LPS) and via complement deposition. The Ail protein from Yersinia pestis is especially promising in that regard, as it confers resistance to the human innate immune response (Kolodziejek). 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). Sialylation would further reduce complement deposition, as well as antibody opsonization. In other words, both Ail and polysialic acid can be produced in concert with our original FtsZ BioBrick to create safer minicells in a desired strain of <i>E. coli</i>. As an additional safety precaution, we also decided to use the <i>lpxM</i> mutant strain, which has a reduction in the acyl-group modifications of the conserved Lipid A portion of its LPS. Studies have shown that this variant can reduce E-selectin stimulation and TNF-α production by up to 10,000-fold (Anisimov). Combined, these three safeguards could hence significantly slash the immunogenicity and risks associated with a minicell chassis.
 </p>
-<p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;In conclusion, 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 associated health risks. By combining multiple safety elements with an inherent ease of production and modularity, we hope to showcase minicells' potential as a chassis for widespread therapeutic use and consumption.
+<p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;In conclusion, 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 <i>IpxM</i> mutant modifications we will implement will further enhance their innate safety, lowering associated health risks. By combining multiple safety elements with an inherent ease of production and modularity, we hope to showcase minicells' potential as a chassis for widespread therapeutic use and consumption.
 </p>