Team:Virginia

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Preliminary Description:

Uneven cellular division in bacteria can yield a normal parent cell and a smaller “minicell”. At 400 nm, minicells are smaller than regular bacteria. They lack chromosomal DNA, which means they cannot replicate and cause infection. On the other hand, they still retain and express plasmid genes, which allow them to carry out a variety of functions (Mugridge). Additionally, minicells maintain a proton gradient and have membrane stability, while also containing proteins from the parent bacteria (Frazer). Since they retain plasma membrane components, these cells can be coupled with antibodies for highly-specific receptor targeting (MacDiarmid). These unique qualities make minicells a viable and safer alternative to comparable chassis such as live E. coli and liposomes.

Minicells offer great promise for synthetic biology. To harness this potential, we aim to create a standard BioBrick that induces minicell formation by E. coli cells, and to design additional safeguards to make our chassis as safe as possible. Research has shown that overproducing the tubulin-like FtsZ protein leads to minicell formation (Ward). Accordingly, we designed our basic BioBrick to contain the FtsZ gene under the control of an IPTG-inducible promoter, so that we can simply overexpress FtsZ to produce minicells. We also decided to model the optimal amount of FtsZ overexpression necessary for robust minicell production.

There are numerous precautions that can be implemented to make minicells even safer for medical use. Among these, we have chosen to incorporate the gene for the Ail protein from Yersinia pestis to prevent complement deposition in human serum (Kolodziejek). We have also explored the possibility of sialylating our chassis with sialic acid, which would lower our minicells’ immunogenicity. Both the Ail protein and the sialic acid can then be expressed in concert with the original FtsZ BioBrick to create safer minicells. All of these genes would then finally be transformed into an IpxM strain, which is less immunogenic than regular E. coli due to a mutation in the lipid A of its lipopolysaccharide (Anisimov). Since LPS is a major target of the complement system, the IpxM strain would thus help the minicells avoid an adverse immune response.

Since minicells are inherently safer than live bacteria, they lower the risk of infection, which is especially important for the young, elderly, or immunocompromised. The safeguard modifications we will implement will add to this innate safety, further lowering associated risks and enhancing minicells’ usability as a safe chassis for therapeutic use.

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.

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 </p>
-<p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Minicells offer great promise for synthetic biology. To harness this potential, we aim to create a standard BioBrick that induces minicell formation by E. coli cells, and designed additional safeguards to make our chassis as safe as possible. Research has shown that overproducing the tubulin-like FtsZ protein leads to minicell formation (Ward). Accordingly, we designed our basic BioBrick to contain the FtsZ gene under the control of an IPTG-inducible promoter, so that we can simply overexpress FtsZ to produce minicells. We also decided to model the optimal amount of FtsZ overexpression necessary for robust minicell production.
+<p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Minicells offer great promise for synthetic biology. To harness this potential, we aim to create a standard BioBrick that induces minicell formation by E. coli cells, and to design additional safeguards to make our chassis as safe as possible. Research has shown that overproducing the tubulin-like FtsZ protein leads to minicell formation (Ward). Accordingly, we designed our basic BioBrick to contain the FtsZ gene under the control of an IPTG-inducible promoter, so that we can simply overexpress FtsZ to produce minicells. We also decided to model the optimal amount of FtsZ overexpression necessary for robust minicell production.
 </p>
-<p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;There are numerous precautions that can be implemented to make minicells even safer for medical use. Among these, we have chosen to incorporate  the gene for the Ail protein from Yersinia pestis to prevent complement deposition in human serum (Kolodziejek). We have also explored the possibility of sialylation using sialic acid on our chassis, which would lower our minicells’ immunogenicity. Both the Ail protein and the sialic acid can then be expressed in concert with the original FtsZ BioBrick to create safe minicells for use as chassis. All of these genes would then finally be transformed into an IpxM strain, which is safer than regular E.coli due to a mutation in the lipid A of its lipopolysaccharide (Anisimov). Since LPS is a major target of the complement system, the IpxM strain would thus help the minicells avoid an adverse immune response.
+<p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;There are numerous precautions that can be implemented to make minicells even safer for medical use. Among these, we have chosen to incorporate  the gene for the Ail protein from Yersinia pestis to prevent complement deposition in human serum (Kolodziejek). We have also explored the possibility of sialylating our chassis with sialic acid, which would lower our minicells’ immunogenicity. Both the Ail protein and the sialic acid can then be expressed in concert with the original FtsZ BioBrick to create safer minicells. All of these genes would then finally be transformed into an IpxM strain, which is less immunogenic than regular E. coli due to a mutation in the lipid A of its lipopolysaccharide (Anisimov). Since LPS is a major target of the complement system, the IpxM strain would thus help the minicells avoid an adverse immune response.
 </p>